Novel fibroblast growth factor and nucleic acids encoding same

ABSTRACT

The present invention provides FGF-CX, a novel isolated polypeptide, as well as a polynucleotide encoding FGF-CX and antibodies that immunospecifically bind to FGF-CX or any derivative, variant, mutant, or fragment of the FGF-CX polypeptide, polynucleotide or antibody. The invention additionally provides methods in which the FGF-CX polypeptide, polynucleotide and antibody are used in detection and treatment of a broad range of pathological states, as well as other uses.

RELATED APPLICATIONS

[0001] This application claims priority to provisional applications U.S.Ser. No. 60/298,441, filed Jun. 15, 2001, U.S. Ser. No. 60/316,446,filed Aug. 31, 2001, U.S. Ser. No. 60/359,594, filed Feb. 26, 2002 andU.S. Ser. No. 60/______, filed May 31, 2002, to Zhong et al. andentitled “Fibroblast growth factor-20-like proteins, derived peptides,and nucleic acids encoding same” and assigned atty docket no. 15966-557U-E; and is a continuation-in-part application of U.S. Ser. No.09/817,814, which is a continuation-in-part application of U.S. Ser. No.09/609,543, which is a continuation-in-part application of U.S. Ser. No.09/494,585, filed Jan. 31, 2000, which in turn claims priority to U.S.Ser. No. 60/145,899, filed Jul. 27, 1999, and further claims priority toPCT patent application US00/20405, filed Jul. 27, 2000. The contents ofeach of these applications are incorporated herein by reference in theirentireties.

FIELD OF THE INVENTION

[0002] The present invention generally relates to nucleic acids andpolypeptides. The invention relates more particularly to nucleic acidsencoding polypeptides related to a member of the fibroblast growthfactor family.

BACKGROUND OF THE INVENTION

[0003] The fibroblast growth factor (FGF) group of cytokines includes atleast 21 members that regulate diverse cellular functions such asgrowth, survival, apoptosis, motility and differentiation. The FGFfamily of proteins, whose prototypic members include acidic FGF (FGF-1)and basic FGF (FGF-2), bind to four related receptor tyrosine kinases.These molecules transduce signals via high affinity interactions withcell surface tyrosine kinase FGF receptors (FGFRs). These FGF receptorsare expressed on most types of cells in tissue culture. Dimerization ofFGF receptor monomers upon ligand binding has been reported to be arequisite for activation of the kinase domains, leading to receptortrans phosphorylation. FGF receptor-1 (FGFR-1), which shows the broadestexpression pattern of the four FGF receptors, contains at least seventyrosine phosphorylation sites. A number of signal transductionmolecules are affected by binding with different affinities to thesephosphorylation sites.

[0004] In addition to participating in normal growth and development,known FGFs have also been implicated in the generation of pathologicalstates, including cancer. FGFs may contribute to malignancy by directlyenhancing the growth of tumor cells. For example, autocrine growthstimulation through the co-expression of FGF and FGFR in the same cellhas been reported to lead to cellular transformation.

SUMMARY OF THE INVENTION

[0005] The present invention is based, in part, upon the discovery of anucleic acid encoding a novel polypeptide having homology to members ofthe Fibroblast Growth Factor (FGF) family of proteins. Included in theinvention are polynucleotide sequences, which are named Fibroblast GrownFactor-CX (FGF-CX), and the FGF-CX polypeptides encoded by these nucleicacid sequences, and splice variants, SNPS, fragments, homologs, analogs,and derivatives thereof, are claimed in the invention. An example of anFGF-CX nucleic acid is SEQ ID NO: 1, and an example of an FGF-CXpolypeptide is a polypeptide including the amino acid sequence of SEQ IDNO: 2. This amino acid sequence is encoded by the nucleic acid sequenceof SEQ ID NO: 1.

[0006] In one aspect, the invention includes an isolated FGF-CXpolypeptide. In some embodiments, the isolated polypeptide includes theamino acid sequence of SEQ ID NO: 2. In other embodiments, the inventionincludes a variant of SEQ ID NO: 2, in which some amino acids residues,e.g., no more than 1%, 2%, 3%, 5%, 10% or 15% of the amino acidsequences of SEQ ID NO; 2 are changed. In some embodiments, the isolatedFGF-CX polypeptide includes the amino acid sequence of a mature form ofan amino acid sequence given by SEQ ID NO: 2, or a variant of a matureform of an amino acid sequence given by SEQ ID NO: 2. Preferably, nomore than 1%, 2%, 3%, 5%, 10% or 15% of the amino acid sequences of SEQID NO; 2 are changed in the variant of the mature form of the amino acidsequence.

[0007] Also include in the invention is a fragment of an FGF-CXpolypeptide, including fragments of variant FGF-CX polypeptides, matureFGF-CX polypeptides and variants of mature FGF-CX polypeptides, as wellas FGF-CX polypeptides encoded by allelic variants and single nucleotidepolymorphisms of FGF-CX nucleic acids. An example of an FGF-CXpolypeptide is a fragment that includes residues 54-211 of SEQ ID NO: 2or residues 24-211 of SEQ ID NO: 2.

[0008] In another aspect, the invention includes an isolated FGF-CXnucleic acid molecule. The FGF-CX nucleic acid molecule can include asequence encoding any of the FGF-CX polypeptides, variants, or fragmentsdisclosed above, or a complement to any such nucleic acid sequence. Inone embodiment, the sequences include those disclosed in SEQ ID NO: 1.In other embodiments, the FGF-CX nucleic acids include a sequencewherein nucleotides different from those given in SEQ ID NO: 1 may beincorporated. Preferably, no more than 1%, 2%, 3,%, 5%, 10%, 15%, or 20%of the nucleotides are so changed.

[0009] In one embodiment, the nucleic acid encodes a polypeptidefragment that includes residues 54-211 of SEQ ID NO: 2 or residues24-211 of SEQ ID NO: 2. The nucleic acid can include, e.g., nucleotides163-633 of SEQ ID NO: 1 or nucleotides 70-633 of SEQ ID NO: 1.

[0010] In other embodiments, the invention includes fragments orcomplements of these nucleic acid sequences. Vectors and cellsincorporating FGF-CX nucleic acids re also included in the invention.

[0011] The invention also includes antibodies that bindimmunospecifically to any of the FGF-CX polypeptides described herein.The FGF-CX antibodies in various embodiments include, e.g., polyclonalantibodies, monoclonal antibodies, humanized antibodies and/or humanantibodies.

[0012] The invention additionally provides pharmaceutical compositionsthat include a FGF-CX polypeptide, a FGF-CX nucleic acid or an FGF-CXantibody of the invention. Also included in the invention are kits thatinclude, e.g., a FGF-CX polypeptide, a FGF-CX nucleic acid or a FGF-CXantibody.

[0013] Several methods are included in the invention. For example, amethod is disclosed for determining the presence or amount of a FGF-CXpolypeptide of the invention in a sample. The method includes contactingthe sample with a FGF-CX antibody that binds immunospecifically to thepolypeptide; and determining the presence or amount of antibody bound tosaid polypeptide, such that the antibody indicates the presence oramount of polypeptide in the sample.

[0014] Similarly, the invention discloses a method for determining thepresence or amount of a FGF-CX nucleic acid molecule in a sample. Themethod includes contacting the sample with a probe that binds to thenucleic acid molecule; and determining the presence or amount of theprobe bound to the nucleic acid molecule, such that the probe indicatesthe presence or amount of the FGF-CX nucleic acid molecule in thesample.

[0015] Also provided by the invention is a method for identifying anagent that binds to a FGF-CX polypeptide. The method includesdetermining whether a candidate substance binds to a FGF-CX polypeptide.Binding of a candidate substance indicates the agent is an FGF-CXpolypeptide binding agent.

[0016] The invention also includes a method for identifying a potentialtherapeutic agent for use in treatment of a pathology. The pathology is,e.g., related to aberrant expression, aberrant processing, or aberrantphysiological interactions of a FGF-CX polypeptide of the invention.This method includes providing a cell which expresses the FGF-CXpolypeptide and has a property or function ascribable to thepolypeptide; contacting the provided cell with a composition comprisinga candidate substance; and determining whether the substance alters theproperty or function ascribable to the polypeptide, in comparison to acontrol cell. Any such substance is identified as a potentialtherapeutic agent. Furthermore, therapeutic agents may be identified bysubjecting any potential therapeutic agent identified in this way toadditional tests to identify a therapeutic agent for use in treating thepathology.

[0017] In some embodiments, the property or function relates to cellgrowth or cell proliferation, and the substance binds to thepolypeptide, thereby modulating an activity of the polypeptide. In someembodiments, the candidate substance has a molecular weight not morethan about 1500 Da. In some embodiments, the candidate substance is anantibody. The invention additionally provides any therapeutic agentidentified using a method such as those described herein.

[0018] Additional important aspects of the invention relate to methodsof treating or preventing a disorder associated with a FGF-CXpolypeptide. The disorder may be characterized by insufficient orineffective growth of a cell or a tissue, or by hyperplasia or neoplasiaof a cell or a tissue. The method includes administering to a subject aFGF-CX polypeptide of the invention, or a FGF-CX nucleic acid of theinvention, or any other Therapeutic of the invention, in an amount andfor a duration sufficient to treat or prevent the disorder in saidsubject. In significant embodiments, the subject is a human.

[0019] The invention also includes a method for screening for amodulator of latency or predisposition to a disorder associated withaberrant expression, aberrant processing, or aberrant physiologicalinteractions of a FGF-CX polypeptide. The method includes providing atest animal that recombinantly expresses the FGF-CX polypeptide of theinvention and is at increased risk for the disorder; administering atest compound to the test animal; measuring an activity of thepolypeptide in the test animal after administering the compound; andcomparing the activity of the FGF-CX polypeptide in the test animal withthe activity of the FGF-CX polypeptide in a control animal notadministered the compound. If there is a change in the activity of thepolypeptide in the test animal relative to the control animal, the testcompound is a modulator of latency of or predisposition to the disorder.

[0020] The invention also provides a method for determining the presenceof or predisposition to a disease associated with altered levels of aFGF-CX polypeptide or of a FGF-CX nucleic acid of the invention in afirst mammalian subject. The method includes measuring the level ofexpression of the polypeptide or the amount of the nucleic acid in asample from the first mammalian subject; and comparing its amount in thesample to its amount present in a control sample from a second mammaliansubject known not to have, or not to be predisposed to, the disease. Analteration in the expression level of the polypeptide or the amount ofthe nucleic acid in the first subject as compared to the control sampleindicates the presence of or predisposition to the disease.

[0021] Also provided by the invention is a method of treating apathological state in a mammal, wherein the pathology is related toaberrant expression, aberrant processing, or aberrant physiologicalinteractions of a FGF-CX polypeptide of the invention. The methodincludes administering to the mammal a polypeptide of the invention inan amount that is sufficient to alleviate the pathological state,wherein the FGF-CX polypeptide is a polypeptide having an amino acidsequence at least 85%, 90%, 95%, 96%, 97%, 98%, or even 99% identical toa polypeptide comprising an amino acid sequence of SEQ ID NO: 2, or abiologically active fragment thereof. In another related method, anantibody of the invention is administered to the mammal.

[0022] In another aspect, the invention, the invention includes a methodof promoting growth of cells in a subject. The method includesadministering to the subject a FGF-CX polypeptide of the invention in anamount and for a duration that are effective to promote cell growth. Insome embodiments, the subject is a human, and the cells whose growth isto be promoted may be chosen from among cells in the vicinity of awound, cells in the vascular system, cells involved in hematopoiesis,cells involved in erythropoiesis, cells in the lining of thegastrointestinal tract, and cells in hair follicles.

[0023] In a further aspect, the invention provides a method ofinhibiting growth of cells in a subject, wherein the growth is relatedto expression of a FGF-CX polypeptide of the invention. This methodincludes administering to the subject a composition that inhibits growthof the cells. In a highly important embodiment, the composition includesan antibody or another therapeutic agent of the invention.Significantly, the subject is a human, and the cells whose growth is tobe inhibited are chosen from among transformed cells, hyperplasticcells, tumor cells, and neoplastic cells.

[0024] In a still further aspect, the invention provides method oftreating or preventing or delaying a tissue proliferation-associateddisorder. The method includes administering to a subject in which suchtreatment or prevention or delay is desired a FGF-CX nucleic acid, aFGF-CX polypeptide, or a FGF-CX antibody in an amount sufficient totreat, prevent, or delay a tissue proliferation-associated disorder inthe subject.

[0025] The tissue proliferation-associated disorders diagnosed, treated,prevented or delayed using the FGF-CX nucleic acid molecules,polypeptides or antibodies can involve epithelial cells, e.g.,fibroblasts and keratinocytes in the anterior eye after surgery. Othertissue proliferation-associated disorder include, e.g., tumors,restenosis, psoriasis, Dupuytren's contracture, diabetic complications,Kaposi sarcoma, and rheumatoid arthritis.

[0026] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

[0027] Other features and advantages of the invention will be apparentfrom the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028]FIG. 1 shows a Western analysis of FGF-CX. Samples from 293 cells(Panel A) or NIH 3T3 cells (Panel B) transiently transfected with theindicated construct were examined by Western analysis using anti-V5antibody. CM=conditioned media, SE=suramin-extracted conditioned media.Molecular mass markers are indicated on the left.

[0029]FIG. 2 shows a Western analysis of FGF-CX protein secreted by 293cells.

[0030]FIG. 3 shows a Western analysis of FGF-CX (SEQ ID NO: 2) proteinexpressed in E. coli cells.

[0031]FIG. 4 present an analysis of the expression of FGF-CX obtained byreal-time quantitative PCR using FGF-CX-specific TaqMan reagents.Results for normalized RNA derived from normal human tissue samples areshown in Panel A, and from tumor cell lines in Panel B. Results obtainedusing tumor tissues obtained directly during surgery are shown in PanelsC and D.

[0032]FIG. 5 displays the biological activity of recombinant FGF-CX asrepresented by its effects on DNA synthesis. Cells were serum-starved,incubated with the indicated factor for 18 hr, and analyzed by a BrdUincorporation assay. Samples were performed in triplicate. Panel A, NIH3T3 mouse fibroblasts. Panel B, CCD-1070 human fibroblasts. Panel C,CCD-1106 human keratinocytes

[0033]FIG. 6 displays the biological activity of recombinant FGF-CX asrepresented by its effects on cell growth. NIH 3T3 cells were incubatedwith serum-free media supplemented with the indicated factor and countedafter 48 hr. Samples were performed in duplicate.

[0034]FIG. 7 presents the biological activity of recombinant FGF-CX asrepresented by its effects on cell morphology. NIH 3T3 cells wereincubated with FGF-CX or control protein for 48 hr and photographed at amagnification of X 25.

[0035]FIG. 8 presents a graph representing the tumorigenic activity ofFGF-CX. NIH 3T3 cells stably transfected with the indicated constructswere injected into the subcutis of athymic nude mice and examined fortumor formation over a two week period. A minimum of 4 animals was usedfor each data point.

[0036]FIG. 9 presents photographs of a control athymic nude mouse and anathymic nude mouse injected subcutis with NIH 3T3 cells stablytransfected with an FGF-CX construct.

[0037]FIG. 10 presents an image of a Coomassie Blue stained SDS-PAGE gelof purified samples of FGF-CX prepared under reducing and nonreducingconditions.

[0038]FIG. 11 provides the results of a dose titration experimentcarried out using 786-0 human renal carcinoma cells. In this experimentincorporation of bromodeoxyuridine induced by varying amounts of FGF-CX(designated in FIG. 21 as 20858) was determined.

[0039]FIG. 13 shows in vitro formation of foci. NIH 3T3 cellstransfected with the indicated constructs were cultured for 2 weeks inDMEM/5% calf serum, stained and photographed. The foci generated by thepIgκ-FGF-20 construct are numerous but small due to overcrowding.

[0040]FIG. 13 shows the results of experiments assessing the receptorbinding specificity of FGF-CX. NIH 3T3 cells were serum-starved,incubated with the indicated growth factor (square=PDGF-BB;triangle=aFGF; circle=FGF-CX) either alone or together with theindicated soluble FGFR, and analyzed by a BrdU incorporation assay.Experiments were performed in triplicate and are represented as thepercent BrdU increase in incorporation of BrdU relative to cellsreceiving the growth factor alone.

[0041]FIG. 14 shows an image of a Coomassie Blue stained SDS-PAGE gel ofthe arginine supernatant obtained when plasmid pET24a-FGF20X-del54-codon was expressed in E. coli strain BL21 (DE3).

[0042]FIG. 15 displays the biological activity of a truncated form ofrecombinant FGF-CX (denoted by (d1-23)FGF20 in the Figure) asrepresented by its effects on DNA synthesis, compared to that of fulllength FGF-CX (denoted FGF20 in the Figure). NIH 3T3 mouse fibroblastswere serum-starved, incubated with the indicated factor for 18 hr, andanalyzed by a BrdU incorporation assay.

DETAILED DESCRIPTION OF THE INVENTION

[0043] This invention is based in part on the discovery of novel FGF-CXnucleic acid sequences, which encode polypeptides that are members ofthe fibroblast growth factor (FGF) family. As used herein thedesignation “FGF-CX” relates to nucleic acids, polynucleotides,proteins, polypeptides, and variants, derivatives and fragments of anyof them, as well as to antibodies that bind immunospecifically to any ofthese classes of compounds.

[0044] Previously described members of the FGF family regulate diversecellular functions such as growth, survival, apoptosis, motility anddifferentiation (Szebenyi & Fallon (1999) Int. Rev. Cytol. 185, 45-106).These molecules transduce signals intracellularly via high affinityinteractions with cell surface tyrosine kinase FGF receptors (FGFRs),four of which have been identified to date (Xu, X., Weinstein, M., Li,C. & Deng, C. (1999) Cell Tissue Res. 296, 33-43; Klint, P. &Claesson-Welsh, L. (1999) Front. Biosci. 4, 165-177). These FGFreceptors are expressed on most types of cells in tissue culture.Dimerization of FGF receptor monomers upon ligand binding has beenreported to be a requisite for activation of the kinase domains, leadingto receptor trans phosphorylation. FGF receptor-1 (FGFR-1), which showsthe broadest expression pattern of the four FGF receptors, contains atleast seven tyrosine phosphorylation sites. A number of signaltransduction molecules are affected by binding with different affinitiesto these phosphorylation sites.

[0045] FGFs also bind, albeit with low affinity, to heparin sulfateproteoglycans (HSPGs) present on most cell surfaces and extracellularmatrices (ECM). Interactions between FGFs and HSPGs serve to stabilizeFGF/FGFR interactions, and to sequester FGFs and protect them fromdegradation (Szebenyi, G. & Fallon, J. F. (1999)). Due to itsgrowth-promoting capabilities, one member of the FGF family, FGF-7, iscurrently in clinical trials for the treatment of chemotherapy-inducedmucositis (Danilenko, D. M. (1999) Toxicol. Pathol. 27, 64-71).

[0046] In addition to participating in normal growth and development,known FGFs have also been implicated in the generation of pathologicalstates, including cancer (Basilico, C & Moscatelli, D. (1992) Adv.Cancer Res. 59, 115-165). FGFs may contribute to malignancy by directlyenhancing the growth of tumor cells. For example, autocrine growthstimulation through the co-expression of FGF and FGFR in the same cellleads to cellular transformation (Matsumoto-Yoshitomi, et al. (1997)Int. J. Cancer 71, 442-450). Likewise, the constitutive activation ofFGFR via mutation or rearrangement leads to uncontrolled proliferation(Lorenzi, et al. (1996) Proc. Natl. Acad. Sci. USA. 93, 8956-8961; Li,et al. (1997) Oncogene 14, 1397-1406). Furthermore, some FGFs areangiogenic (Gerwins, et al. (2000) Crit. Rev. Oncol. Hematol. 34,185-194). Such FGFs may contribute to the tumorigenic process byfacilitating the development of the blood supply needed to sustain tumorgrowth. Not surprisingly, at least one FGF is currently underinvestigation as a potential target for cancer therapy (Gasparini (1999)Drugs 58, 17-38).

[0047] Expression of FGFs and their receptors in the brains of perinataland adult mice has been examined. Messenger RNA all FGF genes, with theexception of FGF-4, is detected in these tissues. FGF-3, FGF-6, FGF-7and FGF-8 genes demonstrate higher expression in the late embryonicstages than in postnatal stages, suggesting that these members areinvolved in the late stages of brain development. In contrast,expression of FGF-1 and FGF-5 increased after birth. In particular,FGF-6 expression in perinatal mice has been reported to be restricted tothe central nervous system and skeletal muscles, with intense signals inthe developing cerebrum in embryos but in cerebellum in 5-day-oldneonates. FGF-receptor (FGFR)-4, a cognate receptor for FGF-6,demonstrate similar spatiotemporal expression, suggesting that FGF-6 andFGFR-4 plays significant roles in the maturation of nervous system as aligand-receptor system. According to Ozawa et al., these resultsstrongly suggest that the various FGFs and their receptors are involvedin the regulation of a variety of developmental processes of brain, suchas proliferation and migration of neuronal progenitor cells, neuronaland glial differentiation, neurite extensions, and synapse formation.

[0048] Other members of the FGF polypeptide family include the FGFreceptor tyrosine kinase (FGFRTK) family and the FGF receptor heparansulfate proteoglycan (FGFRHS) family. These members interact to regulateactive and specific FGFR signal transduction complexes. These regulatoryactivities are diversified throughout a broad range of organs andtissues, and in both normal and tumor tissues, in mammals. Regulatedalternative messenger RNA (mRNA) splicing and combination of variantsubdomains give rise to diversity of FGFRTK monomers. Divalent cationscooperate with the FGFRHS to conformationally restrict FGFRTKtrans-phosphorylation, which causes depression of kinase activity andfacilitates appropriate activation of the FGFR complex by FGF. Forexample, it is known that different point mutations in the FGFRTKcommonly cause craniofacial and skeletal abnormalities of gradedseverity by graded increases in FGF-independent activity of total FGFRcomplexes. Other processes in which FGF family exerts important effectsare liver growth and function and prostate tumor progression.

[0049] Glia-activating factor (GAF), another FGF family member, is aheparin-binding growth factor that was purified from the culturesupernatant of a human glioma cell line. See, Miyamoto et al. 1993, MolCell Biol 13(7): 4251-4259. GAF shows a spectrum of activity slightlydifferent from those of other known growth factors, and is designated asFGF-9. The human FGF-9 cDNA encodes a polypeptide of 208 amino acids.Sequence similarity to other members of the FGF family was estimated tobe around 30%. Two cysteine residues and other consensus sequences foundin other family members were also well conserved in the FGF-9 sequence.FGF-9 was found to have no typical signal sequence in its N terminuslike those in acidic FGF and basic FGF.

[0050] Acidic FGF and basic FGF are known not to be secreted from cellsin a conventional manner. However, FGF-9 was found to be secretedefficiently from cDNA-transfected COS cells despite its lack of atypical signal sequence. It could be detected exclusively in the culturemedium of cells. The secreted protein lacked no amino acid residues atthe N terminus with respect to those predicted by the cDNA sequence,except the initiation methionine. The rat FGF-9 cDNA was also cloned,and the structural analysis indicated that the FGF-9 gene is highlyconserved.

[0051] Using a database search, a human sequence (GenBank AB020858 )from chromosome 8p22-p21.3 was identified that was homologous to XenopusFGF20. See, Kirikoshi et al. Biochem. Biophys. Res. Commun. 274:337-343, 2000, PMID 10913340. PCR primers designed to the human sequencewere used to amplify overlapping FGF20 cDNAs from colon cancer andgastric cancer cell lines. The 3 predicted exons of FGF20 encode adeduced 211 amino acid protein containing an FGF core domain. Thepredicted molecular mass is 23 kD. FGF20 contains a strong hydrophobicregion in the FGF core domain and a weak hydrophobic region at the Nterminus. No N-terminal signal sequence or potential N-glycosylationsites were identified. FGF20 shares 71.6% and 66.2% overall amino acidsequence identity with FGF9 and FGF16, respectively. Kirikoshi et al.(2000) concluded from a phylogenic analysis that FGF20, FGF9, and FGF16form a subfamily among the mammalian FGF family. Using Northern blotanalysis of 20 adult and fetal tissues and 8 cancer cell lines,Kirikoshi et al. (2000) detected a 2.4-kb FGF20 transcript only in acolon cancer cell line, SW480. RT-PCR confirmed the abundant expressionof FGF20 in SW480 cells and demonstrated low levels of expression inhuman fetal brain, fetal liver, fetal kidney, and gastric cancer celllines.

[0052] Kirikoshi et al. (2000) noted that the chromosome 8p22-p21.3region is a frequent site of loss of heterozygosity in human cancer.

[0053] The present invention provides a novel human FGF as well as itscorresponding cDNA. The protein product of this gene has been shown toexhibit growth stimulatory and oncogenic properties. Furthermore,overexpression of the FGF mRNA was noted in certain specific cancer celllines. These observations suggest that the novel FGF may be of use byserving as an excellent target in the treatment of human malignancy.

[0054] The invention also includes mature FGF-CX polypeptides, variantsof mature FGF-CX polypeptides, fragments of mature and mature variantFGF-CX polypeptides, and nucleic acids encoding these polypeptides andfragments. As used herein, a “mature” form of a FGF-CX polypeptide orprotein disclosed in the present invention is the product of a naturallyoccurring polypeptide or precursor form or proprotein. The naturallyoccurring polypeptide, precursor or proprotein includes, by way ofnonlimiting example, the full length gene product, encoded by thecorresponding gene. In some embodiments, the mature form include anFGF-CX polypeptide, precursor or proprotein encoded by an open readingframe described herein. The product “mature” form can arise, e.g., as aresult of one or more naturally occurring processing steps as they maytake place within the cell, or host cell, in which the gene productarises.

[0055] Examples of such processing steps leading to a “mature” form of apolypeptide or protein include the cleavage of the N-terminal methionineresidue encoded by the initiation codon of an open reading frame, or theproteolytic cleavage of a signal peptide or leader sequence. Thus amature form arising from an FGF-CX precursor polypeptide or protein thathas residues 1 to N, where residue I is the N-terminal methionine, wouldhave residues 2 through N remaining after removal of the N-terminalmethionine. Alternatively, a mature form arising from a precursorpolypeptide or protein having residues 1 to N, in which an N-terminalsignal sequence from residue 1 to residue M is cleaved, would have theresidues from residue M+1 residue N remaining. Additionally, a “mature”protein or fragment may arise from a cleavage event other than removalof an initiating methionine or removal of a signal peptide. Further asused herein, a “mature” form of an FGF-CX polypeptide or protein mayarise from a step of post-translational modification other than aproteolytic cleavage event. Such additional processes include, by way ofnon-limiting example, glycosylation, myristoylation or phosphorylation.In general, a mature polypeptide or protein may result from theoperation of only one of these processes, or a combination of any ofthem.

[0056] As used herein, “identical” residues correspond to those residuesin a comparison between two sequences where the equivalent nucleotidebase or amino acid residue in an alignment of two sequences is the sameresidue. Residues are alternatively described as “similar” or “positive”when the comparisons between two sequences in an alignment show thatresidues in an equivalent position in a comparison are either the sameamino acid or a conserved amino acid as defined below.

[0057] Included within the invention are FGF-CX nucleic acids, isolatednucleic acids that encode FGF-CX polypeptide or a portion thereof,FGF-CX polypeptides, vectors containing these nucleic acids, host cellstransformed with the FGF-CX nucleic acids, anti-FGF-CX antibodies, andpharmaceutical compositions. Also disclosed are methods of making FGF-CXpolypeptides, as well as methods of screening, diagnosing, treatingconditions using these compounds, and methods of screening compoundsthat modulate FGF-CX polypeptide activity. Table A below delineates thesequence descriptors that are used throughout the invention. TABLE A SEQID NO SEQUENCE DESCRIPTOR 1 Human FGF-CX nucleotide sequence 2 HumanFGF-CX polypeptide sequence 3 FGF-CX Forward primer 4 FGF-CX Reverseprimer 5 Glia Activating Factor (GAF) 6 Human genomic fragment - bp15927-16214 7 Human genomic fragment - bp 7257-7511 8 Human genomicfragment - bp 9837-9942 9 Human FGF-9 10 Mouse FGF-9 11 Rat FGF-9 12Xenopus FGF-CX 13 Human FGF-CX hydrophobic domain (aa 90-115) 14PSec-V5-His Forward 15 PSec-V5-His Reverse 16 PSETA linker 17 PSETAlinker 18 TaqMan expression analysis forward primer 19 TaqMan expressionanalysis reverse primer 20 TaqMan expression analysis probe 21 Ag8 I (F)TaqMan primer 22 Ag8 I (R) TaqMan primer 23 Ag8 1 (P) TaqMan primer 24FGF-16 polypeptide, GenBank AB009391 25 FGF-CX cDNA 26 CG53135-02 exonlinking primer 27 CG53135-02 exon linking primer 28 CG53135-02 (nt) 29CG53135-02 (aa) 30 CG53135-06 (nt) 31 CG53135-06 (aa) 32 Q9NP95FGFK_Human, Fibroblast Growth Factor-20 33 CG53135-03 (nt) 34 CG53135-03(aa) 35 CG53135-04 (nt) 36 CG53135-04 (aa) 37 CG53135-05 (nt) 38CG53135-05 (aa) 39 CG53135-07 (nt) 40 CG53135-07 (aa) 41 CG53135-08 (nt)42 CG53135-08 (aa) 43 CG53135-09 (nt) 44 CG53135-09 (aa) 45 CG53135-10(nt) 46 CG53135-10 (aa) 47 CG53135 FGF-20-like Protein (nt) 48 CG53135FGF-20-like Protein (aa) 49 CG53135 Variant 13375518 (nt) 50 CG53135Variant 13375518 (aa) 51 CG53135 Variant 13365516 (nt) 52 CG53135Variant 13365516 (aa) 53 CG53135 Variant 13365517 (nt) 54 CG53135Variant 13365517 (aa)

[0058] The FGF-CX nucleic acids and polypeptides, as well as FGF-CXantibodies, therapeutic agents and pharmaceutical compositions discussedherein, are useful, inter alia, in treating tissueproliferation-associated disorders. These tissueproliferation-associated disorders can include disorders affectingepithelial cells, e.g., fibroblasts and keratinocytes in the anterioreye after surgery. Other tissue proliferation-associated disorderinclude, e.g., tumors, restenosis, psoriasis, Dupuytren's contracture,diabetic complications, Kaposi sarcoma, and rheumatoid arthritis.

[0059] Included in the invention is a nucleotide sequence (SEQ ID NO: 1)encoding a novel fibroblast growth factor designated fibroblast growthfactor-20× (FGF-CX ) (see Table 1; SEQ ID NO: 1). This coding sequencewas identified in human genomic DNA sequences. The disclosed DNAsequence has 633 bases that encode a polypeptide predicted to have 211amino acid residues (SEQ ID NO: 2). The predicted molecular weight ofFGF-CX, based on the sequence shown in Table 1 and SEQ ID NO: 2, is23498.4 Da. TABLE 1 Table 1 is a representation of the nucleotidesequence (SEQ ID NO:1) and translated amino acid sequence (SEQ ID NO:2)of a novel FGF-CX polynucleotide and protein of the invention (alsoreferred to as CG53135-01 or Fibroblast Growth Factor AB020858). 1ATGGCTCCCTTAGCCGAAGTCGGGGGCTTTCTGGGCGGCCTGGAGMetAlaProLeuAlaGluValGlyGlyPheLeuGlyGlyLeuGlu 46GGCTTGGGCCAGCAGGTGGGTTCGCATTTCCTGTTGCCTCCTGCCGlyLeuGlyGlnGlnValGlySerHisPheLeuLeuProProAla 91GGGGAGCGGCCGCCGCTGCTGGGCGAGCGCAGGAGCGCGGCGGAGGlyGluArgProProLeuLeuGlyGluArgArgSerAlaAlaGlu 136CGGAGCGCGCGCGGCGGGCCGGGGGCTGCGCAGCTGGCGCACCTGArgSerAlaArgGlyGlyProGlyAlaAlaGlnLeuAlaHisLeu 181CACGGCATCCTGCGCCGCCGGCAGCTCTATTGCCGCACCGGCTTCHisGlyIleLeuArgArgArgGlnLeuTyrCysArgThrGlyPhe 226CACCTGCAGATCCTGCCCGACGGCAGCGTGCAGGGCACCCGGCAGHisLeuGlnIleLeuProAspGlySerValGlnGlyThrArgGln 271GACCACAGCCTCTTCGGTATCTTGGAATTCATCAGTGTGCCAGTGAspHisSerLeuPheGlyIleLeuGluPheIleSerValAlaVal 316GGACTGGTCAGTATTAGAGGTGTGGACAGTGGTCTCTATCTTGGAGlyLeuValSerIleArgGlyValAspSerGlyLeuTyrLeuGly 361ATGAATGACAAAGGAGAACTCTATGGATCAGAGAAACTTACTTCCMetAsnAspLysGlyGluLeuTyrGlySerGluLysLeuThrSer 406GAATGCATCTTTAGGGAGCAGTTTGAAGAGAACTGGTATAACACCGluCysIlePheArgGluGlnPheGluGluAsnTrpTyrAsnThr 451TATTCATCTAACATATATAAACATGGAGACACTGGCCGCAGGTATTyrSerSerAsnIleTyrLysHisGlyAspThrGlyArgArgTyr 496TTTGTGGCACTTAACAAAGACGGAACTCCAAGAGATGGCGCCAGGPheValAlaLeuAsnLysAspGlyThrProArgAspGlyAlaArg 541TCCAAGAGGCATCAGAAATTTACACATTTCTTACCTAGACCAGTGSerLysArgHisGlnLysPheThrHisPheLeuProArgProVal 586GATCCAGAAAGAGTTCCAGAATTGTACAAGGACCTACTGATGTACAspProcluArgValProGluLeuTyrLysAspLeuLeuMetTyr 631 ACT Thr

[0060] The FGF-CX nucleic acid sequence was used as a query nucleotidesequence in a BLASTN search to identify related nucleic acid sequences.The FGF-CX nucleotide sequence has a high similarity to murinefibroblast growth factor 9 (FGF-9) (392 of 543 bases identical, or 72%;GenBank Accession Number S82023) and to human DNA encoding gliaactivating factor (GAP) (385 of 554 bases identical, or 69%; GenBankAccession Number E05822, also termed FGF-9). In addition, FGF-CX wasfound to have a comparable degree of identity (311 of 424 basesidentical, or 73%) to a GAF sequence (SEQ ID NO: 5) disclosed by Naruoet al. in Japanese Patent: JP 1993301893 entitled “Glia-ActivatingFactor And Its Production” (see Table 2). TABLE 2 Table 2 is a BLASTNalignment of the nucleic acid sequence of SEQ ID NO:1 with a FGF-9-likeGlia-Activating factor (GAF) sequence (SEQ ID NO:5). Query: 170TGGCGCACCTGCACGGCATCCTGCGCCGCCGGCAGCTCTATTGCCGCACCGGCTTCCACC 229|||  ||  |  | || || ||  | ||  ||||||| || ||| | || || || |||  Sbjct: 2TGGATCATTTAAAGGGGATTCTCAGGCGGAGGCAGCTATACTGCAGGACTGGATTTCACT 61 Query:230 TGCAGATCCTGCCCGACGGCAGCGTGCAGGGCACCCGGCAGGACCACAGCCTCTTCGGTA 289|  | ||| | ||| | || |   | ||||| ||| || | ||||||||||  || || | Sbjct: 62TAGAAATCTTCCCCAATGGTACTATCCAGGGAACCAGGAAAGACCACAGCCGATTTGGCA 121 Query:290 TCTTGGAATTCATCAGTGTGGCAGTGGGACTGGTCAGTATTAGAGGTGTGGACAGTGGTC 349|  ||||||| |||||| | |||||||| |||||||| ||| |||| ||||||||||| | Sbjct: 122TTCTGGAATTTATCAGTATAGCAGTGGGCCTGGTCAGCATTCGAGGCGTGGACAGTGGAC 181 Query:350 TCTATCTTGGAATGAATGACAAAGGAGAACTCTATGGATCAGAGAAACTTACTTCCGAAT 409|||| || || |||||||| || || || || ||||||||||| ||||| ||    || | Sbjct: 182TCTACCTCGGGATGAATGAGAAGGGGGAGCTCTATGGATCAGAAAAACTAACCCAAGACT 241 Query:410 GCATCTTTAGGGAGCAGTTTGAAGAGAACTGGTATAACACCTATTCATCTAACATATATA 469|  | || || || ||||| ||||| ||||||||||| || || || || ||| |||||| Sbjct: 242GTGTATTCAGAGAACAGTTCGAAGAAAACTGGTATAATACGTACTCGTCAAACCTATATA 301 Query:470 AACATGGAGACACTGGCCGCAGGTATTTTGTGGCACTTAACAAAGACGGAACTCCAAGAG 529| || |  ||||||||  |  | || | ||| ||| | || ||||| || || || |||| Sbjct: 302AGCACGTGGACACTGGAAGGCGATACTATGTTGCATTAAATAAAGATGGGACCCCGAGAG 361 Query:530 ATGGCGCCAGGTCCAAGAGGCATCAGAAATTTACACATTTCTTACCTAGACCAGTGGATC 589| ||  | ||| | ||  |||| |||||||| |||||||| ||||||||||||||||| | Sbjct: 362AAGGGACTAGGACTAAACGGCACCAGAAATTCACACATTTTTTACCTAGACCAGTGGACC 421 Query:590 CAGA 593 | || Sbjct: 422 CCGA 425

[0061] To verify that the open reading frame (ORF) identified by genomicmining was correct, PCR amplification was used to obtain a cDNAcorresponding to the predicted genomic clone. The nucleotide sequence ofthe obtained product precisely matches that of the predicted gene (seeExample 1).

[0062] Table 3 is a BLASTN alignment of the complementary strand of thenucleic acid sequence of SEQ ID NO: 1 with three discontinuous segments(SEQ ID NOs: 6-8 in Tables 3A-3C, respectively) of an extended genomicfragment of human chromosome 8 (GenBank Accession Number AB020858).TABLE 3A Query: 289TACCGAAGAGGCTGTGGTCCTGCCGGGTGCCCTGCACGCTGCCGTCGGGCAGGATCTGCA 230|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct:15927 TACCGAAGAGGCTGTGGTCCTGCCGGGTGCCCTGCACGCTGCCGTCGGGCAGGATCTGCA 15986Query: 229 GGTGGAAGCCGGTGCGGCAATAGAGCTGCCGGCGGCGCAGGATGCCGTGCAGGTGCGCCA170 |||||||||||||||||||||||||||||||||| ||||||||||||||||||||||||| Sbjct:15987 GGTGGAAGCCGGTGCGGCAATAGAGCTGCCGGCG-CGCAGGATGCCGTGCAGGTGCGCCA 16045Query: 169 GCTGCGCAGCCCCCGGCCCGCCGCGCGCGCTCCGCTCCGCCGCGCTCCTGCGCTCGCCCA110 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct:16046 GCTGCGCAGCCCCCGGCCCGCCGCGCGCGCTCCGCTCCGCCGCGCTCCTGCGCTCGCCCA 16105Query: 109 GCAGCGGCGGCCGCTCCCCGGCAGGAGGCAACAGGAAATGCGAACCCACCTGCTGGCCCA50 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct:16106 GCAGCGGCGGCCGCTCCCCGGCAGGAGGCAACAGGAAATGCGAACCCACCTGCTGGCCCA 16165Query: 49 AGCCCTCCAGGCCGCCCAGAAAGCCCCCGACTTCGGCTAAGGGAGCCAT 1||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 16166AGCCCTCCAGGCCGCCCAGAAAGCCCCCGACTTCGGCTAAGGGAGCCAT 16214

[0063] TABLE 3B Query: 633AGTGTACATCAGTAGGTCCTTGTACAATTCTGGAACTCTTTCTGGATCCACTGGTCTAGG 574|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 7257AGTGTACATCAGTAGGTCCTTGTACAATTCTGGAACTCTTTCTGGATCCACTGGTCTAGG 7316 Query:573 TAAGAAATGTGTAAATTTCTGATCCCTCTTGGACCTGGCGCCATCTCTTGGAGTTCCGTC 514|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 7317TAAGAAATGTGTAAATTTCTGATGCCTCTTGGACCTGGCGCCATCTCTTGGAGTTCCGTC 7376 Query:513 TTTGTTAAGTGCCACAAAATACCTGCGGCCAGTGTCTCCATGTTTATATATGTTAGATGA 454|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 7377TTTGTTAACTGCCACAAAATACCTGCGGCCAGTGTCTCCATGTTTATATATGTTAGATCA 7436 Query:453 ATAGGTGTTATACCAGTTCTCTTCAAACTGCTCCCTAAAGATGCATTCGGAAGTAAGTTT 394|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 7437ATAGGTGTTATACCAGTTCTCTTCAAACTGCTCCCTAAAGATGCATTCGGAAGTAAGTTT 7496 Query:393 CTC-TGATCCATAGA 380 ||| ||| | ||| Sbjct: 7497 CTCCTGAAAGAGAGA 7511

[0064] TABLE 3C Query: 391CTGATCCATAGAGTTCTCCTTTGTCATTCATTCCAAGATAGAGACCACTGTCCACACCTC 332|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 9837CTGATCCATAGAGTTCTCCTTTGTCATTCATTCCAAGATAGAGACCACTGTCCACACCTC 9896 Query:331 TAATACTGACCAGTCCCACTGCCACACTGATGAATTCCAAGATACC 286|||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 9897TAATACTGACCAGTCCCACTGCCACACTGATGAATTCCAAGATACC 9942

[0065] The protein encoded by the cDNA is most closely related toXenopus FGF-20× (designated XFGF-CX or XFGF-20× herein), as well as tohuman FGF-9 and human FGF- 16 (80%, 70% and 64% amino acid identity,respectively; see Tables 4 and 5). Based on the strong homology withXFGF-CX, the gene identified in the present disclosure is believed torepresent its human ortholog, and is named FGF-CX herein.

[0066] In addition, amino acid residues that are conserved among FGFfamily members, as indicated by the alignments presented as Table 4 andTable 5, are predicted to be less amenable to alteration. For example,FGF-CX proteins of the present invention can contain at least one domainthat is a typically conserved region in FGF family members, i.e., FGF-9and XFGF-CX proteins, and FGF-CX homologs. As such, these conserveddomains are not likely to be amenable to mutation. Other amino acidresidues, however, (e.g., those that are not conserved or onlysemi-conserved among members of the FGF proteins) may not be asessential for activity and thus are more likely to be amenable toalteration. Black, gray and white represent identical, conserved andnonconserved residues in the alignment, respectively.

[0067] A BLASTP alignment of the first 208 amino acids of the FGF-CXpolypeptide sequence (SEQ ID NO: 2) with a human FGF-9 (SEQ ID NO: 9) isshown in Table 6. See, SWISSPROT Accession Number P31371 forGlia-Activating Factor Precursor (GAF) (Fibroblast Growth Factor-9);Miyamoto et al. 1993 Mol. Cell. Biol. 13:4251-4259; and Naruo et al.1993 J. Biol. Chem. 268:2857-2864. Positive residues include thoseresidues that are either identical (“|”) or have a conservative aminoacid substitution (“+”)in the same relative position of the comparedsequences when aligned, see below. TABLE 6 Table 6 is a BLASTP alignmentof the FGF-CX polypeptide sequence (SEQ ID NO:2) with a human FGF-9 (SEQID NO:9) indicating identical (“|”) and positive (“+”) residues. Query:1 MAPLAEVGGFLGGLEGLGQQVGSHFLLPPAGERPPLLGERRSAAERSARG-GPGAAQLAH 59|||| ||| + |  + +    |+  +||   + | || +    +|      ||    | | Sbjct: 1MAPLGEVGNYFGVQDAV--PFGNVPVLPV--DSPVLLSDHLGQSEAGGLPRGPAVTDLDH 56 Query:60 LHGILRRRQLYCRTGFHLQILPDGSVQGTRQDHSLFGILEFISVAVGLVSIRGVDSGLYL 119| ||||||||||||||||+| |+|++||||+||| ||||||||+|||||||||||||||| Sbjct: 57LKGILRRRQLYCRTGFHLEIFPNGTIQGTRKDHSRFGILEFISIAVGLVSIRGVDSGLYL 116 Query:120 GMNDKGELYGSEKLTSECIFREQFEENWYNTYSSNIYKHGDTGRRYFVALNKDGTPRDGA 179|||||||||||||| ||+||||||||||||||||+||| ||||||+||||||||||+|  Sbjct: 117GMNEKGELYGSEKLTQECVFREQFEENWYNTYSSNLYKHVDTGRRYYVALNKDGTPREGT 176 Query:180 RSKRHQKFTHFLPRPVDPERVPELYKDLL 208 |+||||||||||||||||++|||||||+|Sbjct: 177 RTKRHQKFTNFLPRPVDPDKVPELYKDIL 205

[0068] BLASTX alignments of the first 208 amino acids of the FGF-CXpolypeptide (SEQ ID NO: 2, translated from SEQ ID NO: 1) with the mouseFGF-9 (SEQ ID NO: 10) and rat FGF-9 (SEQ ID NO: 11) sequences are shownin Tables 7 and 8, respectively. See, SWISSPROT Accession Number P54130for Glia-Activating Factor Precursor (GAF) (Fibroblast Growth Factor-9),Santos-Ocampo et al., 1996 J. Biol. Chem. 271:1726-1731, for mouseFGF-9; and SWISSPROT Accession Number P36364 Glia-Activating FactorPrecursor (GAF) (Fibroblast Growth Factor-9) (FGF-9), Miyamoto, 1993Mol. Cell. Biol. 13:4251-4259, for rat FGF-9. TABLE 7 Table 7 is aBLASTX alignment of the FGF-CX polypeptide sequence (SEQ ID NO:2) withmurine FGF-9 (SEQ ID NO:10) indicating identical (“|”) and positive(“+”) residues. Query: 1MAPLAEVGGFLGGLEGLGQQVGSHFLLPPAGERPPLLGERRSAAERSARG-GPGAAQLAH 59|||| ||| + |  + +    |+  +||   + | || +    +|      ||    | | Sbjct: 1MAPLGEVGSYFGVQDAV--PFGNVPVLPV--DSPVLLNDHLGQSEAGGLPRGPAVTDLDH 56 Query:60 LHGILRRRQLYCRTGFHLQILPDGSVQGTRQDHSLFGILEFISVAVGLVSIRGVDSGLYL 119| ||||||||||||||||+| |+|++||||+||| ||||||||+|||||||||||||||| Sbjct: 57LKGILRRRQLYCRTGFHLEIFPNGTIQGTRKDHSRFGILEFISIAVGLVSIRGVDSGLYL 116 Query:120 GMNDKGELYGSEKLTSECIFREQFEENWYNTYSSNIYKHGDTGRRYFVALNKDGTPRDGA 179|||+||||||||||| ||+||||||||||||||||+||| ||||||+||||||||||+|  Sbjct: 117GMNEKGELYGSEKLTQECVFREQFEENWYNTYSSNLYKHVDTGRRYYVALNKDGTPREGT 176 Query:180 RSKRUQKFTHFLPRPVDPERVPELYKDLL 208 |+||||||||||||||||++|||||||+|Sbjct: 177 RTKRHQKFTHFLPRPVDPDKVPELYKDIL 205

[0069] TABLE 8 Table 8 is a BLASTX alignment of the FGF-CX polypeptidesequence (SEQ ID NO:2) with rat FGF-9 (SEQ ID NO:11) indicatingidentical (“|”) and positive (“+”) residues. Query: 1MAPLAEVGGFLGGLEGLGQQVGSHFLLPPAGERPPLLGERRSAAERSARG-GPGAAQLAH 59 ||||||| + | + + |+ +|| + | || + +| || | | Sbjct: 1MAPLGEVGSYFGVQDAV--PFGNVPVLPV--DSPVLLSDHLGQSEAGGLPRGPAVTDLDH 56 Query:60 LHGILRRRQLYCRTGFHLQILPDGSVQGTRQDHSLFGILEFISVAVGLVSIRGVDSGLYL 119 |||||||||||||||||+| |+|++||||+||| ||||||||+|||||||||||||||| Sbjct: 57LKGILRRRQLYCRTGFHLEIFPNGTIQGTRKDHSRFGILEFISIAVGLVSIRGVDSGLYL 116 Query:120 GMNDKGELYGSEKLTSECIFREQFEENWYNTYSSNIYKHGDTGRRYFVALNKDGTPRDGA 179|||+||||||||||| ||+||||||||||||||||+||| ||||||+||||||||||+| Sbjct: 117GMNEKGELYGSEKLTQECVFREQFEENWYNTYSSNLYKHVDTGRRYYVALNKDGTPREGT 176 Query:180 RSKRHQKFTHFLPRPVDPERVPELYKDLL 208 |+||||||||||||||||++|||||||+|Sbjct: 177 RTKRHQKFTHFLPRPVDPDKVPELYKDIL 205

[0070] As indicated by the bars (“|”) in Tables. 6-9, FGF-9 sequences ofall three species have 147 of 208 residues identical with FGF-CX (SEQ IDNO: 2), for an overall sequence identity of 70%. In addition, 170 of 208residues are positive to the sequence of FGF-CX (SEQ ID NO: 2), for anoverall percentage of positive residues of 81%.

[0071] The full length FGF-CX polypeptide (SEQ ID NO: 2) was alsoaligned by BLASTX with Xenopus XFGF-CX (SEQ ID NO: 12). As shown inTable 9, FGF-CX has 170 of 211 (80%) identical residues, and 189 of 211(89%) positive residues compared with Xenopus XFGF-CX. Xenopus XFGF-CXwas obtained recently from a cDNA library prepared at the tailbud stageusing the product of degenerate PCR performed with primers based onmammalian FGF-9s as a probe. See, Koga et al., 1999 Biochem Biophys ResCommun 261(3):756-765. The deduced 208 amino acid sequence of theXFGF-CX open reading frame contains a motif characteristic of the FGFfamily. XFGF-CX has a 73.1% overall similarity to XFGF-9 but differsfrom XFGF-9 in its amino-terminal region (33.3% similarity). Thisresembles the similarity seen for the presently disclosed SEQ ID NO: 2with respect to various mammalian FGF-9 and FGF-16 sequences, includinghuman (see above). See, Tables 4, 5 and 7-9. Table 9 is a BLASTXalignment of the FGF-CX polypeptide sequence (SEQ ID NO: 2) with XenopusXFGF-CX (SEQ ID NO: 12) indicating identical (“|”) and positive (“+”)residues. Query: 1MAPLAEVGGFLGGLEGLGQQVGSHFLLPPAGERPPLLGERRSAAERSARGGPGAAQLAHL 60 |||||+|| |||| + ||| |||||||||| + | |  +  + +|| +|  |  + |+|| Sbjct: 1MAPLADVGTFLGGYDALGQ-VGSHFLLPPAKDSPLLFNDPLAQSERLSRSAP--SDLSHL 57 Query:61 HGILRRRQLYCRTGFHLQILPDGSVQGTRQDHSLFGILEFISVAVGLVSIRGVDSGLYLG 120  ||||||||||||||||||||||+||||||||| ||||||||||+|||||||||+||||| Sbjct: 58QGILRRRQLYCRTGFHLQILPDGNVQGTRQDHSRFGILEFISVAIGLVSIRGVDTGLYLG 117 Query:121 MNDKGELYGSEKLTSECIFREQFEENWYNTYSSNIYKHGDTGRRYFVALNKDGTPRDGAR 180 |||||||+||||||||||||||||||||||||||+|||||+||||||||||||||||| | Sbjct: 118MNDKGELFGSEKLTSECIFREQFEENWYNTYSSNLYKHGDSGRRYFVALNKDGTPRDGTR 177 Query:181 SKRHQKFTHFLPRPVDPERVPELYKDLLMYT 211  +|||||||||||||||||+||||||||+ |+Sbjct: 178 AKRHQKFTHFLPRPVDPEKVPELYKDLMGYS 208

[0072] Additional FGF-20 variants were cloned and identified, asdescribed in the examples. BLASTN and BLASTP analyses were performed fortwo additional variants, specifically CG53135-02 and CG53135-06. Ahigh-scoring match as determined by a BLASTN search of GenBank Composite(no HTG) dated Jun. 12, 2001 using the sequence of the Fibroblast GrowthFactor-20-like gene of the invention. A high-scoring match as determinedby a BLASTP search (versus Non-Redundant Composite dated Jun. 12, 2001)using the sequence of the Fibroblast Growth Factor-20-like protein ofthe invention. Results are shown in Tables 10 and 11, respectively.TABLE 10 BLASTN and BLASTP Results for FGF-20 CG53135-02 variantIdentities Residues/ Similarities for Database Protein/Organism/LengthMatch the Matched Expect Identifier [Patent #, Date] Residues RegionValue AB044277 GENBANK-ID:AB044277 mRNA for FGF-  34 . . 538 495/506(97%) 1.6e−103 20, complete cds - Homo sapiens 262 . . 767 495/506 (97%)1016 bp Q9NP95 SWISSNEW-ACC:Q9NP95 FIBROBLAST 18 . . 179 160/162 (98%)1.6e−93 GROWTH FACTOR-20 (FGF-20) - Homo 50 . . 211 160/162 (98%)sapiens (Human), 211 aa.

[0073] TABLE 11 BLASTN and BLASTP Results for FGF-20 CG53135-06 variantIdentities/ Residues/ Similarities for Database MatchProtein/Organism/Length Match the Matched Expect Identifier [Patent #,Date] Residues Region Value AB044277 GENBANK-ID:AB044277 mRNA for FGF- 34 . . 538 496/506 (98%) 6.9e−104 20, complete cds - Homo sapiens 262 .. 767 496/506 (97%) 1016 bp Q9NP95 SWISSNEW-ACC:Q9NP9S FIBROBLAST 18 . .179 161/162 (99%) 6.3e−94 GROWTH FACTOR-20 (FGF-20) - Homo 50 . . 211161/162 (99%) sapiens (Human), 211 aa.

[0074] The polypeptide sequence in Table 1 (SEQ ID NO: 2) is predictedby the program PSORT to have high probabilities for sorting through themembrane of the endoplasmic reticulum and of the microbody (peroxisome).The CG53135-02 and CG53135-06 variant polypeptides are predicted byPSORT to have a probability of 0.8500 to be in the endoplasmic reticulum(membrane). In alternative embodiments, the CG53135-02 and CG53135-06variant polypeptides are located in the plasma membrane with aprobability of 0.7900, a microbody (peroxisome) with a probability of0.7478 or the mitochondrial inner membrane with a Fib probability of0.100. The CG53135-02 and CG53135-06 variant polypeptides are predictedby the software program INTEGRAL to have a -6.42 likelihood of being atransmembrane domain between amino acid residues 62-78 (60-81). TheFGF-CX polypeptide seems to be a type II (Ncyt Cexo) membrane protein.

[0075] In addition, although it does not have a predicted knowncleavable signal sequence at its N-terminus, a hydropathy plot of theprotein shows that FGF-CX has a prominent hydrophobic segment at aminoacid positions about 90 to about 115 (SEQ ID NO: 13). This singlehydrophobic region is known to be a sorting signal in other members ofthe FGF family. Accordingly, a polypeptide that includes the amino acidsof SEQ ID NO: 13 is useful as a sorting signal, allowing secretionthrough various cellular membranes, such as the endoplasmic reticulum,the Golgi membrane or the plasma membrane. A hydropathy plot of theCG53135-02 and CG53135-06 variant proteins indicates that two prominenthydrophobic segments reside at amino acid positions about 23 to about 60and from amino acid positions about 82 to the end. In variousembodiments, the hydrophobic segments are antigenic and targets forFGF-CX/FGF-20-like protein specific antibodies.

[0076] FGF-CX lacks a classical amino-terminal signal sequence aspredicted by PSORT (Nakai, K & Kanehisa, M. (1992) Genomics 14, 897-911)and SIGNALP (Nielsen, et al. (1997)Protein Eng. 10, 1-6) computeralgorithms, just as found for some of its closest human family members(e.g. FGF-9 and FGF-16). Nonetheless, both FGF-9 and FGF-16 are secreted(Matsumoto-Yoshitomi, et al. (1997) Int. J. Cancer 71, 442-450; Miyake,et al. (1998) Biochem. Biophys. Res. Comm. 243, 148-152; Miyakawa, etal. (1999) J. Biol. Chem. 274, 29352-29357; Revest et al. (2000) J Biol.Chem. 275, 8083-8090). To determine whether FGF-CX is also secreted, thecDNA encoding the full length FGF-CX protein was subcloned into amammalian expression vector designated pFGF-CX. The protein expressedwhen human embryonic kidney 293 cells are transfected with this vectoris found in the conditioned medium, and exhibits a band detected by anantibody to a C-terminal V5 epitope, with an apparent molecular weightin a Western blot of ˜27 kDa (FIG. 1, Example 7). An additional portionof the expressed protein is released from sequestration on the 293 cellsby treatment with a substance that inhibits interaction with heparinsulfate proteoglycan (HSPG). The protein released in this way alsoexhibits a similar Western blot pattern (FIG. 1). Similarly when theprotein is expressed in HEK293 cells from a recombinant plasmidincorporating an Ig Kappa signal sequence, a band is detected by Westernblot with an apparent molecular weight of approximately 34 kDa (FIG. 2,Example 5).

[0077] ClustalW multiple protein alignments (Thompson, et al. (1994)Nucleic Acids Res. 22, 4673-4680) for several vertebrate FGF-likeproteins, including the FGF-CX of the present invention, are shown inFIGS. 4 and 5. The three mammalian proteins (SEQ ID NOs: 9-11) resembleeach other very closely but differ considerably from the FGF-CX proteinof the present invention (SEQ ID NO: 2). Also, the Xenopus XFGF-CX (SEQID NO: 12) and the sequence of SEQ ID NO: 2 resemble each other moreclosely than those of FGF-9. The internal hydrophobic domain involved inFGF-9 secretion (see, e.g., Miyakawa, et al. (1999) J. Biol. Chem. 274,29352-29357) spans residues 95-120 of the FGF-9 sequence. Software fordetermining a hydropathy plot of FGF-CX are well known in the art,including, for example, the Kyte Doolittle, and other algorithms furtherdescribed below.

[0078] The expression of XFGF-20 and of Xenopus FGF-9 are distinct fromeach other. XFGF-20 mRNA is expressed in diploid cells, in embryos atand after the blastula stage, and specifically in the stomach and testisof adults; whereas XFGF-9 mRNA is expressed maternally in eggs and inmany adult tissues. Koga et al., above. Correct expression of XFGF-20during gastrulation appears to be required for the formation of normalhead structures in Xenopus laevis. When XFGF-20 mRNA was overexpressedin early embryos, gastrulation was abnormal and development of anteriorstructures was suppressed. See, Koga et al., above. In such embryos,expression of the Xbra transcript, among those tested, was suppressedduring gastrulation, indicating that expression of the Xbra genemediates XFGF-CX effects. See, Koga et al., above.

[0079] The expression patterns of the related XFGF-9 polypeptide inproliferating tissues, (including, e.g., ova, testis, stomach, andmultiple tissues in the maternal frog), suggests a role for XFGF-20 inthe maintenance of tissues that normally undergo regeneration in afunctioning organism.

[0080] It is shown in Example 8 that FGF-CX mRNA is expressed in normalcerebellum, as well as in several human tumor cell lines includingcarcinomas of the lung, stomach and colon but not in the correspondingnormal tissues. The lack of FGF-CX expression in normal lung, stomachand colon, and its presence in tumor lines from these tissues, indicatesthat these cancer cell lines apparently overexpress FGF-CX in aninappropriate fashion. The chromosomal region to which FGF-CX maps iscommonly altered in colorectal, lung and gastric carcinomas (Emi, et al.(1992) Cancer Res. 52, 5368-5372; Baffa, et al. (2000) Clin. Cancer Res.6, 1372-1377). It is possible that the establishment of an FGF-CX-drivenautocrine growth loop in these cells contributes to their initialtumorigenic conversion and/or to their subsequent expansion. Thisscenario is supported by the finding that the generation of anFGF-CX-driven autocrine loop in NIH 3T3 cells activates theirtumorigenic potential (see Example 11). It is also possible that FGF-CXsecretion by tumor cells stimulates their in vivo growth via paracrineeffects on stromal cells.

[0081] Expression of heterologous FGF-CX in NIH 3T3 cells is found toinduce their transformation and tumorigenicity (see Example 11). Theseeffects are mediated by both native FGF-CX (construct pFGF-CX) andFGF-CX expressed with a heterologous Igκ signal sequence at itsamino-terminus (construct pIgκ-FGF-CX). However, it should be noted thatpIgκ-FGF-CX is more oncogenically active than pFGF-CX, as evidenced byits greater in vitro transforming ability (data not shown) and in vivotumorigenicity (FIG. 8). The superior oncogenicity of pIgκ-FGF-CXrelative to pFGF-CX is likely due to the fact that pIgκ-FGF-CX producessignificantly more secreted FGF-CX protein than does pFGF-CX in NIH 3T3cells (FIG. 1B).

[0082] Like FGF-CX, other FGFs have been shown to transform cellsfollowing ectopic expression, and in some cases the blockade of FGFsignaling has been shown to suppress cell transformation(Matsumoto-Yoshitomi, et al. (1997) Int. J. Cancer 71, 442-450; Li, etal. (1 994) Mol. Cell. Biol. 14, 7660-7669).

[0083] Based on the properties of FGF-CX described herein, as well as onthe similarities with the effects found for related FGF proteins, it isbelieved that FGF-CX plays an important role in human malignancy. Forthese reasons, the FGF-CX polypeptides, nucleic acids and antibodiesdisclosed herein are useful in methods of diagnosing the presence oramounts of these compositions, in screening for and identifyingtherapeutic agents related to FGF-CX-associated pathologies, and inmethods of treatment of various kinds of malignancy.

[0084] FGF-CX Nucleic Acids

[0085] The nucleic acids of the invention include those that encode aFGF-CX or FGF-CX-like protein. Among these nucleic acids is the nucleicacid whose sequence is provided in Table 1 and SEQ ID NO: 1, or afragment thereof. The FGF-CX nucleic acid can have the nucleotidesequence of a genomic FGF-CX nucleic acid, or of a cDNA. Additionally,the invention includes mutant or variant nucleic acids of SEQ ID NO: 1,or a fragment thereof, any of whose bases may be changed from thecorresponding base shown in Table 1 while still encoding a protein thatmaintains its FGF-CX -like activities and physiological functions. Theinvention further includes the complement of the nucleic acid sequenceof SEQ ID NO: 1, including fragments, derivatives, analogs and homologthereof. Examples of the complementary strand of portions of FGF-CX areshown in Table 3. The invention additionally includes nucleic acids ornucleic acid fragments, or complements thereto, whose structures includechemical modifications.

[0086] One aspect of the invention pertains to isolated nucleic acidmolecules that encode FGF-CX proteins or biologically active portionsthereof. Also included are nucleic acid fragments sufficient for use ashybridization probes to identify FGF-CX-encoding nucleic acids (e.g.,FGF-CX mRNA) and fragments for use as polymerase chain reaction (PCR)primers for the amplification or mutation of FGF-CX nucleic acidmolecules. As used herein, the term “nucleic acid molecule” is intendedto include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules(e.g., mRNA), analogs of the DNA or RNA generated using nucleotideanalogs, and derivatives, fragments and homologs thereof. The nucleicacid molecule can be single-stranded or double-stranded, but preferablyis double-stranded DNA.

[0087] “Probes” refer to nucleic acid sequences of variable length,preferably between at least about 10 nucleotides (nt), 100 nt, or asmany as about, e.g., 6,000 nt, depending on use. Probes are used in thedetection of identical, similar, or complementary nucleic acidsequences. Longer length probes are usually obtained from a natural orrecombinant source, are highly specific and much slower to hybridizethan oligomers. Probes may be single- or double-stranded and designed tohave specificity in PCR, membrane-based hybridization technologies, orELISA-like technologies.

[0088] An “isolated” nucleic acid molecule is one that is separated fromother nucleic acid molecules that are present in the natural source ofthe nucleic acid. Examples of isolated nucleic acid molecules include,but are not limited to, recombinant DNA molecules contained in a vector,recombinant DNA molecules maintained in a heterologous host cell,partially or substantially purified nucleic acid molecules, andsynthetic DNA or RNA molecules. Preferably, an “isolated” nucleic acidis free of sequences which naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forexample, in various embodiments, the isolated FGF-CX nucleic acidmolecule can contain less than about 50 kb, 25 kb, 5 kb, 4 kb, 3 kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flankthe nucleic acid molecule in genomic DNA of the cell from which thenucleic acid is derived. Moreover, an “isolated” nucleic acid molecule,such as a cDNA molecule, can be substantially free of other cellularmaterial or culture medium when produced by recombinant techniques, orof chemical precursors or other chemicals when chemically synthesized.

[0089] A nucleic acid molecule of the present invention, e.g., a nucleicacid molecule having the nucleotide sequence of SEQ ID NO: 1, or acomplement of any of this nucleotide sequence, can be isolated usingstandard molecular biology techniques and the sequence informationprovided herein. Using all or a portion of the nucleic acid sequence ofSEQ ID NO: 1 as a hybridization probe, FGF-CX nucleic acid sequences canbe isolated using standard hybridization and cloning techniques (e.g.,as described in Sambrook et al., eds., MOLECULAR CLONING: A LABORATORYMANUAL 2^(nd) Ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989; and Ausubel, et al., eds., CURRENT PROTOCOLS INMOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1993.)

[0090] A nucleic acid of the invention can be amplified using cDNA, mRNAor alternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to FGF-CX nucleotidesequences can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

[0091] As used herein, the term “oligonucleotide” refers to a series oflinked nucleotide residues, which oligonucleotide has a sufficientnumber of nucleotide bases to be used in a PCR reaction. A shortoligonucleotide sequence may be based on, or designed from, a genomic orcDNA sequence and is used to amplify, confirm, or reveal the presence ofan identical, similar or complementary DNA or RNA in a particular cellor tissue. Oligonucleotides comprise portions of a nucleic acid sequencehaving about 10 nt, 50 nt, or 100 nt in length, preferably about 15 ntto 30 nt in length. In one embodiment, an oligonucleotide comprising anucleic acid molecule less than 100 nt in length would further compriseat lease 6 contiguous nucleotides of SEQ ID NO: 1, or a complementthereof. Oligonucleotides may be chemically synthesized and may be usedas probes.

[0092] In another embodiment, an isolated nucleic acid molecule of theinvention comprises a nucleic acid molecule that is a complement of thenucleotide sequence shown in SEQ ID NO: 1. In another embodiment, anisolated nucleic acid molecule of the invention comprises a nucleic acidmolecule that is a complement of the nucleotide sequence shown in SEQ IDNO: 1, or a portion of this nucleotide sequence. A nucleic acid moleculethat is complementary to the nucleotide sequence shown in SEQ ID NO: 1is one that is sufficiently complementary to the nucleotide sequenceshown in SEQ ID NO: 1 that it can hydrogen bond with little or nomismatches to the nucleotide sequence shown in SEQ ID NO: 1, therebyforming a stable duplex.

[0093] As used herein, the term “complementary” refers to Watson-Crickor Hoogsteen base pairing between nucleotides units of a nucleic acidmolecule, and the term “binding” means the physical or chemicalinteraction between two polypeptides or compounds or associatedpolypeptides or compounds or combinations thereof. Binding includesionic, non-ionic, van der Waals, hydrophobic interactions, etc. Aphysical interaction can be either direct or indirect. Indirectinteractions may be through or due to the effects of another polypeptideor compound. Direct binding refers to interactions that do not takeplace through, or due to, the effect of another polypeptide or compound,but instead are without other substantial chemical intermediates.

[0094] Moreover, the nucleic acid molecule of the invention can compriseonly a portion of the nucleic acid sequence of SEQ ID NO: 1, e.g., afragment that can be used as a probe or primer, or a fragment encoding abiologically active portion of FGF-CX. Fragments provided herein aredefined as sequences of at least 6 (contiguous) nucleic acids or atleast 4 (contiguous) amino acids, a length sufficient to allow forspecific hybridization in the case of nucleic acids or for specificrecognition of an epitope in the case of amino acids, respectively, andare at most some portion less than a full length sequence. Fragments maybe derived from any contiguous portion of a nucleic acid or amino acidsequence of choice. Derivatives are nucleic acid sequences or amino acidsequences formed from the native compounds either directly or bymodification or partial substitution. Analogs are nucleic acid sequencesor amino acid sequences that have a structure similar to, but notidentical to, the native compound but differs from it in respect tocertain components or side chains. Analogs may be synthetic or from adifferent evolutionary origin and may have a similar or oppositemetabolic activity compared to wild type.

[0095] Derivatives and analogs may be fall length or other than fulllength, if the derivative or analog contains a modified nucleic acid oramino acid, as described below. Derivatives or analogs of the nucleicacids or proteins of the invention include, but are not limited to,molecules comprising regions that are substantially homologous to thenucleic acids or proteins of the invention, in various embodiments, byat least about 70%, 80%, 85%, 90%, 95%, 98%, or even 99% identity (witha preferred identity of 80-99%) over a nucleic acid or amino acidsequence of identical size or when compared to an aligned sequence inwhich the alignment is done by a computer homology program known in theart, or whose encoding nucleic acid is capable of hybridizing to thecomplement of a sequence encoding the aforementioned proteins understringent, moderately stringent, or low stringent conditions. See e.gAusubel, et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley &Sons, New York, N.Y., 1993, and below. An exemplary program is the Gapprogram (Wisconsin Sequence Analysis Package, Version 8 for UNIX,Genetics Computer Group, University Research Park, Madison, Wis.) usingthe default settings, which uses the algorithm of Smith and Waterman(Adv. Appl. Math., 1981, 2: 482-489, which is incorporated herein byreference in its entirety).

[0096] A “homologous nucleic acid sequence” or “homologous amino acidsequence,” or variations thereof, refer to sequences characterized by ahomology at the nucleotide level or amino acid level as discussed above.Homologous nucleotide sequences encode those sequences coding forisoforms of FGF-CX polypeptide. Isoforms can be expressed in differenttissues of the same organism as a result of, for example, alternativesplicing of RNA. Alternatively, isoforms can be encoded by differentgenes. In the present invention, homologous nucleotide sequences includenucleotide sequences encoding for a FGF-CX polypeptide of species otherthan humans, including, but not limited to, mammals, and thus caninclude, e.g., mouse, rat, rabbit, dog, cat cow, horse, and otherorganisms. Homologous nucleotide sequences also include, but are notlimited to, naturally occurring allelic variations and mutations of thenucleotide sequences set forth herein. A homologous nucleotide sequencedoes not, however, include the nucleotide sequence encoding human FGF-CXprotein. Homologous nucleic acid sequences include those nucleic acidsequences that encode conservative amino acid substitutions (see below)in SEQ ID NO: 2, as well as a polypeptide having FGF-CX activity.Biological activities of the FGF-CX proteins are described below. Ahomologous amino acid sequence does not encode the amino acid sequenceof a human FGF-CX polypeptide.

[0097] The nucleotide sequence determined from the cloning of the humanFGF-CX gene allows for the generation of probes and primers designed foruse in identifying and/or cloning FGF-CX homologues in other cell types,e.g., from other tissues, as well as FGF-CX homologues from othermammals. The probe/primer typically comprises a substantially purifiedoligonucleotide. The oligonucleotide typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400 or moreconsecutive sense strand nucleotide sequence of SEQ ID NO: 1; or ananti-sense strand nucleotide sequence of SEQ ID NO: 1; or of a naturallyoccurring mutant of SEQ ID NO: 1.

[0098] Probes based on the human FGF-CX nucleotide sequence can be usedto detect transcripts or genomic sequences encoding the same orhomologous proteins. In various embodiments, the probe further comprisesa label group attached thereto, e.g., the label group can be aradioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.Such probes can be used as a part of a diagnostic test kit foridentifying cells or tissue which misexpress a FGF-CX protein, such asby measuring a level of a FGF-CX-encoding nucleic acid in a sample ofcells from a subject e.g., detecting FGF-CX mRNA levels or determiningwhether a genomic FGF-CX gene has been mutated or deleted.

[0099] “A polypeptide having a biologically active portion of FGF-CX”refers to polypeptides exhibiting activity similar, but not necessarilyidentical to, an activity of a polypeptide of the present invention,including mature forms, as measured in a particular biological assay,with or without dose dependency. A nucleic acid fragment encoding a“biologically active portion of FGF-CX” can be prepared by isolating aportion of SEQ ID NO: 1, that encodes a polypeptide having a FGF-CXbiological activity (biological activities of the FGF-CX proteins aredescribed below), expressing the encoded portion of FGF-CX protein(e.g., by recombinant expression in vitro) and assessing the activity ofthe encoded portion of FGF-CX. For example, a nucleic acid fragmentencoding a biologically active portion of FGF-CX can optionally includean ATP-binding domain. In another embodiment, a nucleic acid fragmentencoding a biologically active portion of FGF-CX includes one or moreregions.

[0100] FGF-CX Variants

[0101] The invention further encompasses nucleic acid molecules thatdiffer from the nucleotide sequences shown in FIG. 1 due to degeneracyof the genetic code. These nucleic acids thus encode the same FGF-CXprotein as that encoded by the nucleotide sequence shown in SEQ IDNO: 1. In another embodiment, an isolated nucleic acid molecule of theinvention has a nucleotide sequence encoding a protein having an aminoacid sequence shown in SEQ ID NO: 2.

[0102] In addition to the human FGF-CX nucleotide sequence shown in SEQID NO: 1, it will be appreciated by those skilled in the art that DNAsequence polymorphisms that lead to changes in the amino acid sequencesof FGF-CX may exist within a population (e.g., the human population).Such genetic polymorphism in the FGF-CX gene may exist among individualswithin a population due to natural allelic variation. As used herein,the terms “gene” and “recombinant gene” refer to nucleic acid moleculescomprising an open reading frame encoding a FGF-CX protein, preferably amammalian FGF-CX protein. Such natural allelic variations can typicallyresult in 1-5% variance in the nucleotide sequence of the FGF-CX gene.Any and all such nucleotide variations and resulting amino acidpolymorphisms in FGF-CX that are the result of natural allelic variationand that do not alter the functional activity of FGF-CX are intended tobe within the scope of the invention.

[0103] Moreover, nucleic acid molecules encoding FGF-CX proteins fromother species, and thus that have a nucleotide sequence that differsfrom the human sequence of SEQ ID NO: 1, are intended to be within thescope of the invention. Nucleic acid molecules corresponding to naturalallelic variants and homologues of the FGF-CX cDNAs of the invention canbe isolated based on their homology to the human FGF-CX nucleic acidsdisclosed herein using the human cDNAs, or a portion thereof, as ahybridization probe according to standard hybridization techniques understringent hybridization conditions. For example, a soluble human FGF-CXcDNA can be isolated based on its homology to human membrane-boundFGF-CX. Likewise, a membrane-bound human FGF-CX cDNA can be isolatedbased on its homology to soluble human FGF-CX.

[0104] Accordingly, in another embodiment, an isolated nucleic acidmolecule of the invention is at least 6 nucleotides in length andhybridizes under stringent conditions to the nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO: 1. In anotherembodiment, the nucleic acid is at least 10, 25, 50, 100, 250, 500 or750 nucleotides in length. In another embodiment, an isolated nucleicacid molecule of the invention hybridizes to the coding region. As usedherein, the term “hybridizes under stringent conditions” is intended todescribe conditions for hybridization and washing under which nucleotidesequences at least 60% homologous to each other typically remainhybridized to each other.

[0105] Homologs (i e., nucleic acids encoding FGF-CX proteins derivedfrom species other than human) or other related sequences (e.g.,paralogs) can be obtained by low, moderate or high stringencyhybridization with all or a portion of the particular human sequence asa probe using methods well known in the art for nucleic acidhybridization and cloning.

[0106] As used herein, the phrase “stringent hybridization conditions”refers to conditions under which a probe, primer or oligonucleotide willhybridize to its target sequence, but to no other sequences. Stringentconditions are sequence-dependent and will be different in differentcircumstances. Longer sequences hybridize specifically at highertemperatures than shorter sequences. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (Tm) forthe specific sequence at a defined ionic strength and pH. The Tm is thetemperature (under defined ionic strength, pH and nucleic acidconcentration) at which 50% of the probes complementary to the targetsequence hybridize to the target sequence at equilibrium. Since thetarget sequences are generally present at excess, at Tm, 50% of theprobes are occupied at equilibrium. Typically, stringent conditions willbe those in which the salt concentration is less than about 1.0 M sodiumion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0to 8.3 and the temperature is at least about 30° C. for short probes,primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about60° C. for longer probes, primers and oligonucleotides. Stringentconditions may also be achieved with the addition of destabilizingagents, such as formamide.

[0107] Stringent conditions such as described above are known to thoseskilled in the art and can be found in CURRENT PROTOCOLS IN MOLECULARBIOLOGY, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Preferably, theconditions are such that sequences at least about 65%, 70%, 75%, 85%,90%, 95%, 98%, or 99% homologous to each other typically remainhybridized to each other. A non-limiting example of stringenthybridization conditions is hybridization in a high salt buffercomprising 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02%Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65° C.This hybridization is followed by one or more washes in 0.2×SSC, 0.01%BSA at 50° C. An isolated nucleic acid molecule of the invention thathybridizes under stringent conditions to the sequence of SEQ ID NO: 1corresponds to a naturally occurring nucleic acid molecule. As usedherein, a “naturally-occurring” nucleic acid molecule refers to an RNAor DNA molecule having a nucleotide sequence that occurs in nature(e.g., encodes a natural protein).

[0108] Homologs (i.e., nucleic acids encoding FGF-CX proteins derivedfrom species other than human) or other related sequences (e.g.,paralogs) can be obtained by low, moderate or high stringencyhybridization with all or a portion of the particular human sequence asa probe using methods well known in the art for nucleic acidhybridization and cloning.

[0109] In a second embodiment, a nucleic acid sequence that ishybridizable to the nucleic acid molecule comprising the nucleotidesequence of SEQ ID NO: 1, or fragments, analogs or derivatives thereof,under conditions of moderate stringency is provided. A non-limitingexample of moderate stringency hybridization conditions arehybridization in 6×SSC, 5×Denhardt's solution, 0.5% SDS and 100 mg/mldenatured salmon sperm DNA at 55° C., followed by one or more washes in1×SSC, 0.1% SDS at 37° C. Other conditions of moderate stringency thatmay be used are well known in the art. See, e.g., Ausubel et al. (eds.),1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, andKriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL,Stockton Press, NY.

[0110] In a third embodiment, a nucleic acid that is hybridizable to thenucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, or fragments, analogs or derivatives thereof, under conditions of lowstringency, is provided. A non-limiting example of low stringencyhybridization conditions are hybridization in 35% formamide, 5×SSC, 50mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100mg/ml denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40°C., followed by one or more washes in 2×SSC, 25 mM Tris-HCl (pH 7.4), 5mM EDTA, and 0.1% SDS at 50° C. Other conditions of low stringency thatmay be used are well known in the art (e.g., as employed forcross-species hybridizations). See, e.g., Ausubel et al. (eds.), 1993,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, andKriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL,Stockton Press, NY; Shilo and Weinberg, 1981, Proc Natl Acad Sci USA 78:6789-6792.

[0111] Conservative mutations

[0112] In addition to naturally-occurring allelic variants of the FGF-CXsequence that may exist in the population, the skilled artisan willfurther appreciate that changes can be introduced by mutation into thenucleotide sequence of SEQ ID NO: 1, thereby leading to changes in theamino acid sequence of the encoded FGF-CX protein, without altering thefunctional ability of the FGF-CX protein. For example, nucleotidesubstitutions leading to amino acid substitutions at “non-essential”amino acid residues can be made in the sequence of SEQ ID NO: 1. A“non-essential” amino acid residue is a residue that can be altered fromthe wild-type sequence of FGF-CX without altering the biologicalactivity, whereas an “essential” amino acid residue is required forbiological activity. For example, amino acid residues that are conservedamong the FGF-CX proteins of the present invention, are predicted to beparticularly unamenable to alteration.

[0113] Another aspect of the invention pertains to nucleic acidmolecules encoding FGF-CX proteins that contain changes in amino acidresidues that are not essential for activity. Such FGF-CX proteinsdiffer in amino acid sequence from SEQ ID NO: 2, yet retain biologicalactivity. In one embodiment, the isolated nucleic acid moleculecomprises a nucleotide sequence encoding a protein, wherein the proteincomprises an amino acid sequence at least about 75% homologous to theamino acid sequence of SEQ ID NO: 2. Preferably, the protein encoded bythe nucleic acid is at least about 80% homologous to SEQ ID NO: 2, morepreferably at least about 90%, 95%, 98%, and most preferably at leastabout 99% homologous to SEQ ID NO: 2.

[0114] An isolated nucleic acid molecule encoding a FGF-CX proteinhomologous to the protein of SEQ ID NO: 2 can be created by introducingone or more nucleotide substitutions, additions or deletions into thenucleotide sequence of SEQ ID NO: 1, such that one or more amino acidsubstitutions, additions or deletions are introduced into the encodedprotein.

[0115] Mutations can be introduced into SEQ ID NO: 1 by standardtechniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Preferably, conservative amino acid substitutions are madeat one or more predicted non-essential amino acid residues. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. Certain amino acids have side chains with morethan one classifiable characteristic. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,tryptophan, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tyrosine,tryptophan), beta-branched side chains (e.g., threonine, valine,isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine,tryptophan, histidine). Thus, a predicted nonessential amino acidresidue in a growth factor is replaced with another amino acid residuefrom the same side chain family. Alternatively, in another embodiment,mutations can be introduced randomly along all or part of a growthfactor coding sequence, such as by saturation mutagenesis, and theresultant mutants can be screened for growth factor biological activityto identify mutants that retain activity. Following mutagenesis of SEQID NOS: 1 and 3 the encoded protein can be expressed by any recombinanttechnology known in the art and the activity of the protein can bedetermined.

[0116] In an important embodiment, a mutant FGF-CX protein can beassayed for (1) the ability to form protein:protein interactions withother FGF-CX proteins, other cell-surface proteins, or biologicallyactive portions thereof, (2) complex formation between a mutant FGF-CXprotein and a FGF-CX receptor; (3) the ability of a mutant FGF-CXprotein to bind to an intracellular target protein or biologicallyactive portion thereof; (e.g., avidin proteins); or (4) the ability tospecifically bind an anti-FGF-CX protein antibody.

[0117] Antisense

[0118] Another aspect of the invention pertains to isolated antisensenucleic acid molecules that are hybridizable to or complementary to thenucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, or fragments, analogs or derivatives thereof. An “antisense” nucleicacid comprises a nucleotide sequence that is complementary to a “sense”nucleic acid encoding a protein, e.g., complementary to the codingstrand of a double-stranded cDNA molecule or complementary to an mRNAsequence. In specific aspects, antisense nucleic acid molecules areprovided that comprise a sequence complementary to at least about 10,25, 50, 100, 250 or 500 nucleotides or an entire FGF-CX coding strand,or to only a portion thereof. Nucleic acid molecules encoding fragments,homologs, derivatives and analogs of a FGF-CX protein of SEQ ID NO: 2 orantisense nucleic acids complementary to a FGF-CX nucleic acid sequenceof SEQ ID NO: 1 are additionally provided.

[0119] In one embodiment, an antisense nucleic acid molecule isantisense to a “coding region” of the coding strand of a nucleotidesequence encoding FGF-CX. The term “coding region” refers to the regionof the nucleotide sequence comprising codons which are translated intoamino acid residues (e.g., the protein coding region of human FGF-CXcorresponds to SEQ ID NO: 2). In another embodiment, the antisensenucleic acid molecule is antisense to a “noncoding region” of the codingstrand of a nucleotide sequence encoding FGF-CX. The term “noncodingregion” refers to 5′ and 3′ sequences which flank the coding region thatare not translated into amino acids (i.e., also referred to as 5′ and 3′untranslated regions).

[0120] Given the coding strand sequences encoding FGF-CX disclosedherein (e.g., SEQ ID NO: 1 ), antisense nucleic acids of the inventioncan be designed according to the rules of Watson and Crick or Hoogsteenbase pairing. The antisense nucleic acid molecule can be complementaryto the entire coding region of FGF-CX mRNA, but more preferably is anoligonucleotide that is antisense to only a portion of the coding ornoncoding region of FGF-CX mRNA. For example, the antisenseoligonucleotide can be complementary to the region surrounding thetranslation start site of FGF-CX mRNA. An antisense oligonucleotide canbe, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50nucleotides in length. An antisense nucleic acid of the invention can beconstructed using chemical synthesis or enzymatic ligation reactionsusing procedures known in the art. For example, an antisense nucleicacid (e.g., an antisense oligonucleotide) can be chemically synthesizedusing naturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed between theantisense and sense nucleic acids, e.g., phosphorothioate derivativesand acridine substituted nucleotides can be used.

[0121] Examples of modified nucleotides that can be used to generate theantisense nucleic acid include: 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

[0122] The antisense nucleic acid molecules of the invention aretypically administered to a subject or generated in situ such that theyhybridize with or bind to cellular mRNA and/or genomic DNA encoding aFGF-CX protein to thereby inhibit expression of the protein, e.g., byinhibiting transcription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule that binds toDNA duplexes, through specific interactions in the major groove of thedouble helix. An example of a route of administration of antisensenucleic acid molecules of the invention includes direct injection at atissue site. Alternatively, antisense nucleic acid molecules can bemodified to target selected cells and then administered systemically.For example, for systemic administration, antisense molecules can bemodified such that they specifically bind to receptors or antigensexpressed on a selected cell surface, e.g., by linking the antisensenucleic acid molecules to peptides or antibodies that bind to cellsurface receptors or antigens. The antisense nucleic acid molecules canalso be delivered to cells using the vectors described herein. Toachieve sufficient intracellular concentrations of antisense molecules,vector constructs in which the antisense nucleic acid molecule is placedunder the control of a strong pol II or pol III promoter are preferred.

[0123] In yet another embodiment, the antisense nucleic acid molecule ofthe invention is an α-anomeric nucleic acid molecule. An (x-anomericnucleic acid molecule forms specific double-stranded hybrids withcomplementary RNA in which, contrary to the usual β-units, the strandsrun parallel to each other (Gaultier et al. (1987) Nucleic Acids Res 15:6625-6641). The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res 15:6131-6148) or a chimeric RNA -DNA analogue (Inoue et al. (1987) FEBSLett 215: 327-330).

[0124] Ribozymes and PNA moieties

[0125] Such modifications include, by way of nonlimiting example,modified bases, and nucleic acids whose sugar phosphate backbones aremodified or derivatized. These modifications are carried out at least inpart to enhance the chemical stability of the modified nucleic acid,such that they may be used, for example, as antisense binding nucleicacids in therapeutic applications in a subject.

[0126] In still another embodiment, an antisense nucleic acid of theinvention is a ribozyme. Ribozymes are catalytic RNA molecules withribonuclease activity that are capable of cleaving a single-strandednucleic acid, such as an mRNA, to which they have a complementaryregion. Thus, ribozymes (e.g., hammerhead ribozymes (described inHaselhoff and Gerlach (1988) Nature 334:585-591)) can be used tocatalytically cleave FGF-CX mRNA transcripts to thereby inhibittranslation of FGF-CX mRNA. A ribozyme having specificity for aFGF-CX-encoding nucleic acid can be designed based upon the nucleotidesequence of a FGF-CX DNA disclosed herein (i.e., SEQ ID NO: 1). Forexample, a derivative of a Tetrahymena L-19 IVS RNA can be constructedin which the nucleotide sequence of the active site is complementary tothe nucleotide sequence to be cleaved in a FGF-CX-encoding mRNA. See,e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No.5,116,742. Alternatively, FGF-CX mRNA can be used to select a catalyticRNA having a specific ribonuclease activity from a pool of RNAmolecules. See, e.g., Bartel et al., (1993) Science 261:1411-1418.

[0127] Alternatively, FGF-CX gene expression can be inhibited bytargeting nucleotide sequences complementary to the regulatory region ofthe FGF-CX (e.g., the FGF-CX promoter and/or enhancers) to form triplehelical structures that prevent transcription of the FGF-CX gene intarget cells. See generally, Helene. (1991) Anticancer Drug Des. 6:569-84; Helene. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher(1992) Bioassays 14: 807-15.

[0128] In various embodiments, the nucleic acids of FGF-CX can bemodified at the base moiety, sugar moiety or phosphate backbone toimprove, e.g., the stability, hybridization, or solubility of themolecule. For example, the deoxyribose phosphate backbone of the nucleicacids can be modified to generate peptide nucleic acids (see Hyrup etal. (1996) Bioorg Med Chem 4: 5-23). As used herein, the terms “peptidenucleic acids” or “PNAs” refer to nucleic acid mimics, e.g., DNA mimics,in which the deoxyribose phosphate backbone is replaced by apseudopeptide backbone and only the four natural nucleobases areretained. The neutral backbone of PNAs has been shown to allow forspecific hybridization to DNA and RNA under conditions of low ionicstrength. The synthesis of PNA oligomers can be performed using A5standard solid phase peptide synthesis protocols as described in Hyrupet al. (1996) above; Perry-O'Keefe et al. (1996) PNAS 93: 14670-675.

[0129] PNAs of FGF-CX can be used in therapeutic and diagnosticapplications. For example, PNAs can be used as antisense or antigeneagents for sequence-specific modulation of gene expression by, e.g.,inducing transcription or translation arrest or inhibiting replication.PNAs of FGF-CX can also be used, e.g., in the analysis of single basepair mutations in a gene by, e.g., PNA directed PCR clamping; asartificial restriction enzymes when used in combination with otherenzymes, e.g., S1 nucleases (Hyrup B. (1996) above); or as probes orprimers for DNA sequence and hybridization (Hyrup et al. (1996), above;Perry-O'Keefe (1996), above).

[0130] In another embodiment, PNAs of FGF-CX can be modified, e.g., toenhance their stability or cellular uptake, by attaching lipophilic orother helper groups to PNA, by the formation of PNA-DNA chimeras, or bythe use of liposomes or other techniques of drug delivery known in theart. For example, PNA-DNA chimeras of FGF-CX can be generated that maycombine the advantageous properties of PNA and DNA. Such chimeras allowDNA recognition enzymes, e.g., RNase H and DNA polymerases, to interactwith the DNA portion while the PNA portion would provide high bindingaffinity and specificity. PNA-DNA chimeras can be linked using linkersof appropriate lengths selected in terms of base stacking, number ofbonds between the nucleobases, and orientation (Hyrup (1996) above). Thesynthesis of PNA-DNA chimeras can be performed as described in Hyrup(1996) above and Finn et al. (1996) Nucl Acids Res 24: 3357-63. Forexample, a DNA chain can be synthesized on a solid support usingstandard phosphoramidite coupling chemistry, and modified nucleosideanalogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidinephosphoramidite, can be used between the PNA and the 5′ end of DNA (Maget al. (1989) Nucl Acid Res 17: 5973-88). PNA monomers are then coupledin a stepwise manner to produce a chimeric molecule with a 5′ PNAsegment and a 3′ DNA segment (Finn et al. (1996) above). Alternatively,chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNAsegment. See, Petersen et al. (1975) Bioorg Med Chem Lett 5: 1119-11124.

[0131] In other embodiments, the oligonucleotide may include otherappended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci.U.S.A. 86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci.84:648-652; PCT Publication No. W088/09810) or the blood-brain barrier(see, e.g., PCT Publication No. W089/10134). In addition,oligonucleotides can be modified with hybridization triggered cleavageagents (See, e.g., Krol et al., 1988, Bio Techniques 6:958-976) orintercalating agents. (See, e.g., Zon, 1988, Pharm. Res. 5: 539-549). Tothis end, the oligonucleotide may be conjugated to another molecule,e.g., a peptide, a hybridization triggered cross-linking agent, atransport agent, a hybridization-triggered cleavage agent, etc.

[0132] FGF-CX polypeptides

[0133] The novel protein of the invention includes the FGF-CX-likeprotein whose sequence is provided in Table 1 (SEQ ID NO: 2). Theinvention also includes a mutant or variant protein any of whoseresidues may be changed from the corresponding residue shown in Table 1while still encoding a protein that maintains its FGF-CX-like activitiesand physiological functions, or a functional fragment thereof. In themutant or variant protein, up to 20% or more of the residues may be sochanged.

[0134] In general, an FGF-CX -like variant that preserves FGF-CX-likefunction includes any variant in which residues at a particular positionin the sequence have been substituted by other amino acids, and furtherinclude the possibility of inserting an additional residue or residuesbetween two residues of the parent protein as well as the possibility ofdeleting one or more residues from the parent sequence. Any amino acidsubstitution, insertion, or deletion is encompassed by the invention. Infavorable circumstances, the substitution is a conservative substitutionas defined above. Furthermore, without limiting the scope of theinvention, the following positions in Table 12 (using the numberingprovided in SEQ ID NO: 2) may be substituted as indicated, such that amutant or variant protein may include one or more than one of thesubstitutions indicated. The suggested substitutions do not limit therange of possible substitutions that may be made at a given position.TABLE 12. POSITION POSSIBLE SUBSTITUTION 6: Glu to Asp 9: Gly to Ser,Thr, or Asn 10: Phe to Tyr 11: Leu to Phe or Ile 15: Glu to Asp 16: Glyto Ala 17: Leu to Ile or Val 19: Gin may be deleted 21: Val to Phe orIle 31: Gly to Lys, Arg, Ser, or Ala 33: Arg to Lys or Ser 35: Pro toLeu or Val 38: Gly to Asn or Ser 39: Glu to Asp 40: Arg to Lys, His, orPro 42: Ser to Thr, Ala, or Gly 43: Ala to Gln, Asn, or Ser 48: Ala toSer or Gly 51: Gly to Ala 53: Gly to Ala or deleted 54: Ala to Gly, Val,or deleted 55: Ala to Ser or Thr 56: Gln to Asp, Glu, or Asn 58: Ala toSet, Thr, Asn, Gln, Asp, or Glu 61: His to Gln, Asn, Lys, or Arg 78: Glnto Asn, Glu, or Asp 80: Leu to Phe or Ile 82: Asp to Glu, Asn, or Gln84: Ser to Asn, Thr, or Gln 85: Val to Ile 90: Gln to Asn or Lys 103:Val to Ile 115: Ser to Thr 123: Asp to Glu 128: Tyr to Phe 135: Set toThr, Gln, or Asn 138: Ile to Val or Leu 155: Ile to Len 159: Gly to Valor Ala 161: Thr to Ser 166: Phe to Tyr 177: Asp to Glu 181: Ser to Alaor Thr 198: Glu to Asp 199: Arg to Lys 207: Leu to Ile or Val 209: Metto any residue 211: Thr to Ser

[0135] One aspect of the invention pertains to isolated FGF-CX proteins,and biologically active portions thereof, or derivatives, fragments,analogs or homologs thereof. Also provided are polypeptide fragmentssuitable for use as immunogens to raise anti-FGF-CX antibodies. In oneembodiment, native FGF-CX proteins can be isolated from cells or tissuesources by an appropriate purification scheme using standard proteinpurification techniques. In another embodiment, FGF-CX proteins areproduced by recombinant DNA techniques. Alternative to recombinantexpression, a FGF-CX protein or polypeptide can be synthesizedchemically using standard peptide synthesis techniques.

[0136] An “isolated” or “purified” protein or biologically activeportion thereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theFGF-CX protein is derived, or substantially free from chemicalprecursors or other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations ofFGF-CX protein in which the protein is separated from cellularcomponents of the cells from which it is isolated or recombinantlyproduced. In one embodiment, the language “substantially free ofcellular material” includes preparations of FGF-CX protein having lessthan about 30% (by dry weight) of non-FGF-CX protein (also referred toherein as a “contaminating protein”), more preferably less than about20% of non-FGF-CX protein, still more preferably less than about 10% ofnon-FGF-CX protein, and most preferably less than about 5% non-FGF-CXprotein. When the FGF-CX protein or biologically active portion thereofis recombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,more preferably less than about 10%, and most preferably less than about5% of the volume of the protein preparation.

[0137] The language “substantially free of chemical precursors or otherchemicals” includes preparations of FGF-CX protein in which the proteinis separated from chemical precursors or other chemicals that areinvolved in the synthesis of the protein. In one embodiment, thelanguage “substantially free of chemical precursors or other chemicals”includes preparations of FGF-CX protein having less than about 30% (bydry weight) of chemical precursors or non-FGF-CX chemicals, morepreferably less than about 20% chemical precursors or non-FGF-CXchemicals, still more preferably less than about 10% chemical precursorsor non-FGF-CX chemicals, and most preferably less than about 5% chemicalprecursors or non-FGF-CX chemicals.

[0138] Biologically active portions of a FGF-CX protein include peptidescomprising amino acid sequences sufficiently homologous to or derivedfrom the amino acid sequence of the FGF-CX protein, e.g., the amino acidsequence shown in SEQ ID NO: 2 that include fewer amino acids than thefull length FGF-CX proteins, and exhibit at least one activity of aFGF-CX protein. Typically, biologically active portions comprise adomain or motif with at least one activity of the FGF-CX protein. Abiologically active portion of a FGF-CX protein can be a polypeptidewhich is, for example, 10, 25, 50, 100 or more amino acids in length.

[0139] A biologically active portion of a FGF-CX protein of the presentinvention may contain at least one of the above-identified domainssubstantially conserved between the FGF family of proteins. Moreover,other biologically active portions, in which other regions of theprotein are deleted, can be prepared by recombinant techniques andevaluated for one or more of the functional activities of a nativeFGF-CX protein.

[0140] In an embodiment, the FGF-CX protein has an amino acid sequenceshown in SEQ ID NO: 2 In other embodiments, the FGF-CX protein issubstantially homologous to SEQ ID NO:2 and retains the functionalactivity of the protein of SEQ ID NO: 2, yet differs in amino acidsequence due to natural allelic variation or mutagenesis, as describedin detail below. Accordingly, in another embodiment, the FGF-CX proteinis a protein that comprises an amino acid sequence at least about 45%homologous to the amino acid sequence of SEQ ID NO: 2 and retains thefunctional activity of the FGF-CX proteins of SEQ ID NO: 2. In anotherembodiment, the FGF-CX is a protein that contains an amino acid sequenceat least about 45% homologous, and more preferably about 55, 65, 70, 75,80, 85, 90, 95, 98 or even 99% homologous to the amino acid sequence ofSEQ ID NO: 2 and retains the functional activity of the FGF-CX proteinsof the corresponding polypeptide having the sequence of SEQ ID NO: 2.

[0141] Determining homology between two or more sequences

[0142] To determine the percent homology of two amino acid sequences orof two nucleic acids, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in either of the sequences beingcompared for optimal alignment between the sequences). The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules arehomologous at that position (i.e., as used herein amino acid or nucleicacid “homology” is equivalent to amino acid or nucleic acid “identity”).

[0143] The nucleic acid sequence homology may be determined as thedegree of identity between two sequences. The homology may be determinedusing computer programs known in the art, such as GAP software providedin the GCG program package. See, Needleman and Wunsch 1970 J Mol Biol48: 443-453. Using GCG GAP software with the following settings fornucleic acid sequence comparison: GAP creation penalty of 5.0 and GAPextension penalty of 0.3, the coding region of the analogous nucleicacid sequences referred to above exhibits a degree of identitypreferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, withthe CDS (encoding) part of the DNA sequence shown in SEQ ID NO: 1.

[0144] The term “sequence identity” refers to the degree to which twopolynucleotide or polypeptide sequences are identical on aresidue-by-residue basis over a particular region of comparison. Theterm “percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over that region of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I, in the case of nucleic acids) occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the region ofcomparison (i.e., the window size), and multiplying the result by 100 toyield the percentage of sequence identity. The term “substantialidentity” as used herein denotes a characteristic of a polynucleotidesequence, wherein the polynucleotide comprises a sequence that has atleast 80 percent sequence identity, preferably at least 85 percentidentity and often 90 to 95 percent sequence identity, more usually atleast 99 percent sequence identity as compared to a reference sequenceover a comparison region. The term “percentage of positive residues” iscalculated by comparing two optimally aligned sequences over that regionof comparison, determining the number of positions at which theidentical and conservative amino acid substitutions, as defined above,occur in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the region of comparison (i.e., the window size), andmultiplying the result by 100 to yield the percentage of positiveresidues.

[0145] Chimeric and fusion proteins

[0146] The invention also provides FGF-CX chimeric or fusion proteins.As used herein, a FGF-CX “chimeric protein” or “fusion protein”comprises a FGF-CX polypeptide operatively linked to a non-FGF-CXpolypeptide. A “FGF-CX polypeptide” refers to a polypeptide having anamino acid sequence corresponding to FGF-CX, whereas a “non-FGF-CXpolypeptide” refers to a polypeptide having an amino acid sequencecorresponding to a protein that is not substantially homologous to theFGF-CX protein, e.g., a protein that is different from the FGF-CXprotein and that is derived from the same or a different organism.Within a FGF-CX fusion protein the FGF-CX polypeptide can correspond toall or a portion of a FGF-CX protein. In one embodiment, a FGF-CX fusionprotein comprises at least one biologically active portion of a FGF-CXprotein. In another embodiment, a FGF-CX fusion protein comprises atleast two biologically active portions of a FGF-CX protein. Within thefusion protein, the term “operatively linked” is intended to indicatethat the FGF-CX polypeptide and the non-FGF-CX polypeptide are fusedin-frame to each other. The non-FGF-CX polypeptide can be fused to theN-terminus or C-terminus of the FGF-CX polypeptide.

[0147] For example, in one embodiment a FGF-CX fusion protein comprisesa FGF-CX polypeptide operably linked to the extracellular domain of asecond protein. Such fusion proteins can be further utilized inscreening assays for compounds that modulate FGF-CX activity (suchassays are described in detail below).

[0148] In another embodiment, the fusion protein is a GST-FGF-CX fusionprotein in which the FGF-CX sequences are fused to the C-terminus of theGST (i.e., glutathione S-transferase) sequences. Such fusion proteinscan facilitate the purification of recombinant FGF-CX.

[0149] In yet another embodiment, the fusion protein is a FGF-CX proteincontaining a heterologous signal sequence at its N-terminus. Forexample, the native FGF-CX signal sequence (i.e., amino acids 1 to 20 ofSEQ ID NO: 2 ) can be removed and replaced with a signal sequence fromanother protein. In certain host cells (e.g., mammalian host cells),expression and/or secretion of FGF-CX can be increased through use of aheterologous signal sequence.

[0150] In another embodiment, the fusion protein is aFGF-CX-immunoglobulin fusion protein in which the FGF-CX sequencescomprising one or more domains are fused to sequences derived from amember of the immunoglobulin protein family. The FGF-CX-immunoglobulinfusion proteins of the invention can be incorporated into pharmaceuticalcompositions and administered to a subject to inhibit an interactionbetween a FGF-CX ligand and a FGF-CX protein on the surface of a cell,to thereby suppress FGF-CX-mediated signal transduction in vivo. In onenonlimiting example, a contemplated FGF-CX ligand of the invention isthe FGF-CX receptor. The FGF-CX-immunoglobulin fusion proteins can beused to affect the bioavailability of a FGF-CX cognate ligand.Inhibition of the FGF-CX ligand/FGF-CX interaction may be usefultherapeutically for both the treatment of proliferative anddifferentiative disorders, as well as modulating (e.g., promoting orinhibiting) cell survival. Moreover, the FGF-CX-immunoglobulin fusionproteins of the invention can be used as immunogens to produceanti-FGF-CX antibodies in a subject, to purify FGF-CX ligands, and inscreening assays to identify molecules that inhibit the interaction ofFGF-CX with a FGF-CX ligand.

[0151] A FGF-CX chimeric or fusion protein of the invention can beproduced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, e.g., byemploying blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers that give rise tocomplementary overhangs between two consecutive gene fragments that cansubsequently be annealed and reamplified to generate a chimeric genesequence (see, for example, Ausubel et al. (eds.) CURRENT PROTOCOLS INMOLECULAR BIOLOGY, John Wiley & Sons, 1992). Moreover, many expressionvectors are commercially available that already encode a fusion moiety(e.g., a GST polypeptide). A FGF-CX-encoding nucleic acid can be clonedinto such an expression vector such that the fusion moiety is linkedin-frame to the FGF-CX protein.

[0152] FGF-CX Agonists and Antagonists

[0153] The present invention also pertains to variants of the FGF-CXproteins that function as either FGF-CX agonists (mimetics) or as FGF-CXantagonists. Variants of the FGF-CX protein can be generated bymutagenesis, e.g., discrete point mutation or truncation of the FGF-CXprotein. An agonist of the FGF-CX protein can retain substantially thesame, or a subset of, the biological activities of the naturallyoccurring form of the FGF-CX protein. An antagonist of the FGF-CXprotein can inhibit one or more of the activities of the naturallyoccurring form of the FGF-CX protein by, for example, competitivelybinding to a downstream or upstream member of a cellular signalingcascade which includes the FGF-CX protein. Thus, specific biologicaleffects can be elicited by treatment with a variant of limited function.In one embodiment, treatment of a subject with a variant having a subsetof the biological activities of the naturally occurring form of theprotein has fewer side effects in a subject relative to treatment withthe naturally occurring form of the FGF-CX proteins.

[0154] Variants of the FGF-CX protein that function as either FGF-CXagonists (mimetics) or as FGF-CX antagonists can be identified byscreening combinatorial libraries of mutants, e.g., truncation mutants,of the FGF-CX protein for FGF-CX protein agonist or antagonist activity.In one embodiment, a variegated library of FGF-CX variants is generatedby combinatorial mutagenesis at the nucleic acid level and is encoded bya variegated gene library. A variegated library of FGF-CX variants canbe produced by, for example, enzymatically ligating a mixture ofsynthetic oligonucleotides into gene sequences such that a degenerateset of potential FGF-CX sequences is expressible as individualpolypeptides, or alternatively, as a set of larger fusion proteins(e.g., for phage display) containing the set of FGF-CX sequencestherein. There are a variety of methods which can be used to producelibraries of potential FGF-CX variants from a degenerate oligonucleotidesequence. Chemical synthesis of a degenerate gene sequence can beperformed in an automatic DNA synthesizer, and the synthetic gene thenligated into an appropriate expression vector. Use of a degenerate setof genes allows for the provision, in one mixture, of all of thesequences encoding the desired set of potential FGF-CX sequences.Methods for synthesizing degenerate oligonucleotides are known in theart (see, e.g., Narang (1983) Tetrahedron 39:3; Itakura et al. (1984)Annu Rev Biochem 53:323; Itakura et al. (1984) Science 198:1056; Ike etal. (1983)Nucl Acid Res 11:477.

[0155] Polypeptide libraries

[0156] In addition, libraries of fragments of the FGF-CX protein codingsequence can be used to generate a variegated population of FGF-CXfragments for screening and subsequent selection of variants of a FGF-CXprotein. In one embodiment, a library of coding sequence fragments canbe generated by treating a double stranded PCR fragment of a FGF-CXcoding sequence with a nuclease under conditions wherein nicking occursonly about once per molecule, denaturing the double stranded DNA,renaturing the DNA to form double stranded DNA that can includesense/antisense pairs from different nicked products, removing singlestranded portions from reformed duplexes by treatment with SI nuclease,and ligating the resulting fragment library into an expression vector.By this method, an expression library can be derived which encodesN-terminal and internal fragments of various sizes of the FGF-CXprotein.

[0157] Several techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations ortruncation, and for screening cDNA libraries for gene products having aselected property. Such techniques are adaptable for rapid screening ofthe gene libraries generated by the combinatorial mutagenesis of FGF-CXproteins. The most widely used techniques, which are amenable to highthroughput analysis, for screening large gene libraries typicallyinclude cloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates isolation of the vectorencoding the gene whose product was detected. Recrusive ensemblemutagenesis (REM), a new technique that enhances the frequency offunctional mutants in the libraries, can be used in combination with thescreening assays to identify FGF-CX variants (Arkin and Yourvan (1992)PNAS 89:7811-7815; Delgrave et al. (1993) Protein Engineering6:327-331).

[0158] Anti-FGF-CX Antibodies

[0159] The term “antibody” as used herein refers to immunoglobulinmolecules and immunologically active portions of immunoglobulin (Ig)molecules, i.e., molecules that contain an antigen binding site thatspecifically binds (immunoreacts with) an antigen. Such antibodiesinclude, but are not limited to, polyclonal, monoclonal, chimeric,single chain, Fab, Fab′ and F(ab′)2 fragments, and an Fab expressionlibrary. In general, antibody molecules obtained from humans relates toany of the classes IgG, IgM, IgA, IgE and IgD, which differ from oneanother by the nature of the heavy chain present in the molecule.Certain classes have subclasses as well, such as IgG1, IgG2, and others.Furthermore, in humans, the light chain may be a kappa chain or a lambdachain. Reference herein to antibodies includes a reference to all suchclasses, subclasses and types of human antibody species.

[0160] An isolated protein of the invention intended to serve as anantigen, or a portion or fragment thereof, can be used as an immunogento generate antibodies that immunospecifically bind the antigen, usingstandard techniques for polyclonal and monoclonal antibody preparation.The full-length protein can be used or, alternatively, the inventionprovides antigenic peptide fragments of the antigen for use asimmunogens. An antigenic peptide fragment comprises at least 6 aminoacid residues of the amino acid sequence of the full length protein,such as an amino acid sequence shown in SEQ ID NO: 2, and encompasses anepitope thereof such that an antibody raised against the peptide forms aspecific immune complex with the full length protein or with anyfragment that contains the epitope. Preferably, the antigenic peptidecomprises at least 10 amino acid residues, or at least 15 amino acidresidues, or at least 20 amino acid residues, or at least 30 amino acidresidues. Preferred epitopes encompassed by the antigenic peptide areregions of the protein that are located on its surface; commonly theseare hydrophilic regions.

[0161] In certain embodiments of the invention, at least one epitopeencompassed by the antigenic peptide is a region of the FGF-CX that islocated on the surface of the protein, e.g., a hydrophilic region. Ahydrophobicity analysis of the human FGF-CX protein sequence willindicate which regions of a FGF-CX polypeptide are particularlyhydrophilic and, therefore, are likely to encode surface residues usefulfor targeting antibody production. As a means for targeting antibodyproduction, hydropathy plots showing regions of hydrophilicity andhydrophobicity may be generated by any method well known in the art,including, for example, the Kyte Doolittle or the Hopp Woods methods,either with or without Fourier transformation. See, e.g., Hopp andWoods, 1981, Proc. Nat. Acad. Sci. USA 78: 3824-3828; Kyte and Doolittle1982, J. Mol. Biol. 157: 105-142, each incorporated herein by referencein their entirety. Antibodies that are specific for one or more domainswithin an antigenic protein, or derivatives, fragments, analogs orhomologs thereof, are also provided herein.

[0162] A protein of the invention, or a derivative, fragment, analog,homolog or ortholog thereof, may be utilized as an immunogen in thegeneration of antibodies that immunospecifically bind these proteincomponents.

[0163] Various procedures known within the art may be used for theproduction of polyclonal or monoclonal antibodies directed against aprotein of the invention, or against derivatives, fragments, analogshomologs or orthologs thereof (see, for example, Antibodies: ALaboratory Manual, Harlow E, and Lane D, 1988, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., incorporated herein byreference). Some of these antibodies are discussed below.

[0164] 1. Polyclonal Antibodies

[0165] For the production of polyclonal antibodies, various suitablehost animals (e.g., rabbit, goat, mouse or other mammal) may beimmunized by one or more injections with the FGF-CX native protein, asynthetic variant thereof, or a derivative of the foregoing. Anappropriate immunogenic preparation can contain, for example, thenaturally occurring immunogenic protein, a chemically synthesizedpolypeptide representing the immunogenic protein, or a recombinantlyexpressed immunogenic protein. Furthermore, the FGF-CX protein may beconjugated to a second protein known to be immunogenic in the mammalbeing immunized. Examples of such immunogenic proteins include but arenot limited to keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, and soybean trypsin inhibitor. The preparation canfurther include an adjuvant. Various adjuvants used to increase theimmunological response include, but are not limited to, Freund's(complete and incomplete), mineral gels (e.g., aluminum hydroxide),surface active substances (e.g., lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, dinitrophenol, etc.), adjuvantsusable in humans such as Bacille Calmette-Guerin and Corynebacteriumparvum, or similar immunostimulatory agents. Additional examples ofadjuvants which can be employed include MPL-TDM adjuvant (monophosphorylLipid A, synthetic trehalose dicorynomycolate).

[0166] The polyclonal antibody molecules directed against theimmunogenic FGF-CX protein can be isolated from the mammal (e.g., fromthe blood) and further purified by well known techniques, such asaffinity chromatography using protein A or protein G, which provideprimarily the IgG fraction of immune serum. Subsequently, oralternatively, the specific antigen which is the target of theimmunoglobulin sought, or an epitope thereof, may be immobilized on acolumn to purify the immune specific antibody by immunoaffinitychromatography. Purification of immunoglobulins is discussed, forexample, by D. Wilkinson (The Scientist, published by The Scientist,Inc., Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000), pp. 25-28).

[0167] 2. Monoclonal Antibodies

[0168] The term “monoclonal antibody” (MAb) or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one molecular species of antibody moleculeconsisting of a unique light chain gene product and a unique heavy chaingene product. In particular, the complementarity determining regions(CDRs) of the monoclonal antibody are identical in all the molecules ofthe population. MAbs thus contain an antigen binding site capable ofimmunoreacting with a particular epitope of the antigen characterized bya unique binding affinity for it.

[0169] Monoclonal antibodies can be prepared using hybridoma methods,such as those described by Kohler and Milstein, Nature, 256:495 (1975).In a hybridoma method, a mouse, hamster, or other appropriate hostanimal, is typically immunized with an immunizing agent to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the immunizing agent. Alternatively, thelymphocytes can be immunized in vitro.

[0170] The immunizing agent will typically include the FGF-CX proteinantigen, a fragment thereof or a fusion protein thereof. Generally,either peripheral blood lymphocytes are used if cells of human originare desired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. The lymphocytes are then fused with animmortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell. See, e.g., Goding,Monoclonal Antibodies: Principles and Practice, Academic Press, (1986)pp. 59-103. Immortalized cell lines are usually transformed mammaliancells, particularly myeloma cells of rodent, bovine and human origin.Usually, rat or mouse myeloma cell lines are employed. The hybridomacells can be cultured in a suitable culture medium that preferablycontains one or more substances that inhibit the growth or survival ofthe unfused, immortalized cells. For example, if the parental cells lackthe enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT orHPRT), the culture medium for the hybridomas typically will includehypoxanthine, aminopterin, and thymidine (“HAT medium”), whichsubstances prevent the growth of HGPRT-deficient cells.

[0171] Preferred immortalized cell lines are those that fuseefficiently, support stable high level expression of antibody by theselected antibody-producing cells, and are sensitive to a medium such asHAT medium. More preferred immortalized cell lines are murine myelomalines, which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies. See, e.g., Kozbor: J. Immunol., 133:3001 (1984);Brodeur et al.: Monoclonal Antibody Production Techniques andApplications, Marcel Dekker, Inc., New York, (1987) pp. 51-63.

[0172] The culture medium in which the hybridoma cells are cultured canthen be assayed for the presence of monoclonal antibodies directedagainst the antigen. Preferably, the binding specificity of monoclonalantibodies produced by the hybridoma cells is determined byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).Such techniques and assays are known in the art. The binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchardanalysis of Munson and Pollard, Anal. Biochem., 107:220 (1980). It is anobjective, especially important in therapeutic applications ofmonoclonal antibodies, to identify antibodies having a high degree ofspecificity and a high binding affinity for the target antigen.

[0173] After the desired hybridoma cells are identified, the clones canbe subcloned by limiting dilution procedures and grown by standardmethods (Goding,1986). Suitable culture media for this purpose include,for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells can be grown in vivo as ascites in amammal.

[0174] The monoclonal antibodies secreted by the subclones can beisolated or purified from the culture medium or ascites fluid byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

[0175] The monoclonal antibodies can also be made by recombinant DNAmethods, such as those described in U.S. Pat. No. 4,816,567. DNAencoding the monoclonal antibodies of the invention can be readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of murine antibodies). The hybridomacells of the invention serve as a preferred source of such DNA. Onceisolated, the DNA can be placed into expression vectors, which are thentransfected into host cells such as simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. The DNA also can be modified, forexample, by substituting the coding sequence for human heavy and lightchain constant domains in place of the homologous murine sequences (U.S.Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide. Such anon-immunoglobulin polypeptide can be substituted for the constantdomains of an antibody of the invention, or can be substituted for thevariable domains of one antigen-combining site of an antibody of theinvention to create a chimeric bivalent antibody.

[0176] 3. Humanized Antibodies

[0177] The antibodies directed against the FGF-CX protein antigens ofthe invention can further comprise humanized antibodies or humanantibodies. These antibodies are suitable for administration to humanswithout engendering an immune response by the human against theadministered immunoglobulin. Humanized forms of antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies)that are principally comprised of the sequence of a humanimmunoglobulin, and contain minimal sequence derived from a non-humanimmunoglobulin. Humanization can be performed following the method ofWinter and co-workers (Jones et al., Nature, 321:522-525 (1986);Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science,239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences forthe corresponding sequences of a human antibody. (See also U.S. Pat. No.5,225,539.) In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies can also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of theframework regions are those of a human immunoglobulin consensussequence. The humanized antibody optimally also will comprise at least aportion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; andPresta, Curr. Op. Struct. Biol., 2:593-596 (1992)).

[0178] 4. Human Antibodies

[0179] Fully human antibodies essentially relate to antibody moleculesin which the entire sequence of both the light chain and the heavychain, including the CDRs, arise from human genes. Such antibodies aretermed “human antibodies”, or “fully human antibodies” herein. Humanmonoclonal antibodies directed against a FGF-CX protein can be preparedby the trioma technique; the human B-cell hybridoma technique (seeKozbor, et al., 1983 Immunol Today 4: 72) and the EBV hybridomatechnique to produce human monoclonal antibodies (see Cole, et al., 1985In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Human monoclonal antibodies may be utilized in the practice ofthe present invention and may be produced by using human hybridomas (seeCote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or bytransforming human B-cells with Epstein Barr Virus in vitro (see Cole,et al., 1985 In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96).

[0180] In addition, human antibodies can also be produced usingadditional techniques, including phage display libraries (Hoogenboom andWinter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991)). Similarly, human antibodies can be made by introducinghuman immunoglobulin loci into transgenic animals, e.g., mice in whichthe endogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al.(Bio/Technology 10, 779-783 (1992)); Lonberg et al. (Nature 368 856-859(1994)); Morrison (Nature 368, 812-13 (1994)); Fishwild et al,( NatureBiotechnology 14, 845-51 (1996)); Neuberger (Nature Biotechnology 14,826 (1996)); and Lonberg and Huszar (Intern. Rev. Immunol. 13 65-93(1995)).

[0181] Human antibodies that specifically bind a FGF-CX protein mayadditionally be produced using transgenic nonhuman animals which aremodified so as to produce fully human antibodies rather than theanimal's endogenous antibodies in response to challenge by an antigen.(See publication WO 94/02602). The endogenous genes encoding the heavyand light immunoglobulin chains in the nonhuman host have beenincapacitated, and active loci encoding human heavy and light chainimmunoglobulins are inserted into the host's genome. The human genes areincorporated, for example, using yeast artificial chromosomes containingthe requisite human DNA segments. An animal which provides all thedesired modifications is then obtained as progeny by crossbreedingintermediate transgenic animals containing fewer than the fullcomplement of the modifications. The preferred embodiment of such anonhuman animal is a mouse, and is termed the XenomouseTM as disclosedin PCT publications WO 96/33735 and WO 96/34096. This animal produces Bcells which secrete fully human immunoglobulins. The antibodies can beobtained directly from the animal after immunization with a FGF-CXimmunogen of interest, as, for example, a preparation of a polyclonalantibody, or alternatively from immortalized B cells derived from theanimal, such as hybridomas producing monoclonal antibodies.Additionally, the genes encoding the immunoglobulins with human variableregions can be recovered and expressed to obtain the antibodiesdirectly, or can be further modified to obtain analogs of antibodiessuch as, for example, single chain Fv molecules.

[0182] An example of a method of producing a nonhuman host, exemplifiedas a mouse, lacking expression of an endogenous immunoglobulin heavychain is disclosed in U.S. Pat. No. 5,939,598. It can be obtained by amethod including deleting the J segment genes from at least oneendogenous heavy chain locus in an embryonic stem cell to preventrearrangement of the locus and to prevent formation of a transcript of arearranged immunoglobulin heavy chain locus, the deletion being effectedby a targeting vector containing a gene encoding a selectable marker;and producing from the embryonic stem cell a transgenic mouse whosesomatic and germ cells contain the gene encoding the selectable marker.

[0183] A method for producing an antibody of interest, such as a humanantibody, is disclosed in U.S. Pat. No. 5,916,771. It includesintroducing an expression vector that contains a nucleotide sequenceencoding a heavy chain into one mammalian host cell in culture,introducing an expression vector containing a nucleotide sequenceencoding a light chain into another mammalian host cell, and fusing thetwo cells to form a hybrid cell. The hybrid cell expresses an antibodycontaining the heavy chain and the light chain.

[0184] In a further improvement on this procedure, a method foridentifying a clinically relevant epitope on an immunogen, and acorrelative method for selecting an antibody that bindsimmunospecifically to the relevant epitope with high affinity, aredisclosed in PCT publication WO 99/53049.

[0185] 5. Fab Fragments and Single Chain Antibodies

[0186] According to the invention, techniques can be adapted for theproduction of single-chain antibodies specific to an antigenic FGF-CXprotein of the invention (see e.g., U.S. Pat. No. 4,946,778). Inaddition, methods can be adapted for the construction of Fab expressionlibraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allowrapid and effective identification of monoclonal Fab fragments with thedesired specificity for a protein or derivatives, fragments, analogs orhomologs thereof. Antibody fragments that contain the idiotypes to aprotein antigen may be produced by techniques known in the artincluding, but not limited to: (i) an F(ab′)2 fragment produced bypepsin digestion of an antibody molecule; (ii) an Fab fragment generatedby reducing the disulfide bridges of an F(ab′)2 fragment; (iii) an Fabfragment generated by the treatment of the antibody molecule with papainand a reducing agent and (iv) Fv fragments.

[0187] 6. Bispecific Antibodies

[0188] Bispecific antibodies are monoclonal, preferably human orhumanized, antibodies that have binding specificities for at least twodifferent antigens. In the present case, one of the bindingspecificities is for an antigenic protein of the invention. The secondbinding target is any other antigen, and advantageously is acell-surface protein or receptor or receptor subunit.

[0189] Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305:537-539 (1983)). Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published May 13, 1993, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

[0190] Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

[0191] According to another approach described in WO 96/27011, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the CH3 region of an antibody constant domain. In this method,one or more small amino acid side chains from the interface of the firstantibody molecule are replaced with larger side chains (e.g. tyrosine ortryptophan). Compensatory “cavities” of identical or similar size to thelarge side chain(s) are created on the interface of the second antibodymolecule by replacing large amino acid side chains with smaller ones(e.g. alanine or threonine). This provides a mechanism for increasingthe yield of the heterodimer over other unwanted end-products such ashomodimers.

[0192] Bispecific antibodies can be prepared as full length antibodiesor antibody fragments (e.g. F(ab′)2 bispecific antibodies). Techniquesfor generating bispecific antibodies from antibody fragments have beendescribed in the literature. For example, bispecific antibodies can beprepared using chemical linkage. Brennan et al., Science 229:81 (1985)describe a procedure wherein intact antibodies are proteolyticallycleaved to generate F(ab′)2 fragments. These fragments are reduced inthe presence of the dithiol complexing agent sodium arsenite tostabilize vicinal dithiols and prevent intermolecular disulfideformation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

[0193] Additionally, Fab′ fragments can be directly recovered from E.coli and chemically coupled to form bispecific antibodies. Shalaby etal., J. Exp. Med. 175:217-225 (1992) describe the production of a fullyhumanized bispecific antibody F(ab′)2 molecule. Each Fab′ fragment wasseparately secreted from E. coli and subjected to directed chemicalcoupling in vitro to form the bispecific antibody. The bispecificantibody thus formed was able to bind to cells overexpressing the ErbB2receptor and normal human T cells, as well as trigger the lytic activityof human cytotoxic lymphocytes against human breast tumor targets.

[0194] Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (VH) connected to a light-chain variabledomain (VL) by a linker which is too short to allow pairing between thetwo domains on the same chain. Accordingly, the VH and VL domains of onefragment are forced to pair with the complementary VL and VH domains ofanother fragment, thereby forming two antigen-binding sites. Anotherstrategy for making bispecific antibody fragments by the use ofsingle-chain Fv (sFv) dimers has also been reported. See, Gruber et al.,J. Immunol. 152:5368 (1994).

[0195] Antibodies with more than two valencies are contemplated. Forexample, trispecific antibodies can be prepared. Tutt et al., J.Immunol. 147:60 (1991).

[0196] Exemplary bispecific antibodies can bind to two differentepitopes, at least one of which originates in the protein antigen of theinvention. Alternatively, an anti-antigenic arm of an immunoglobulinmolecule can be combined with an arm which binds to a triggeringmolecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2,CD3, CD28, or B7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64),FcγRII (CD32) and FcγRIII (CD16) so as to focus cellular defensemechanisms to the cell expressing the particular antigen. Bispecificantibodies can also be used to direct cytotoxic agents to cells whichexpress a particular antigen. These antibodies possess anantigen-binding arm and an arm which binds a cytotoxic agent or aradionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Anotherbispecific antibody of interest binds the protein antigen describedherein and further binds tissue factor (TF).

[0197] 7. Heteroconjugate Antibodies

[0198] Heteroconjugate antibodies are also within the scope of thepresent invention. Heteroconjugate antibodies are composed of twocovalently joined antibodies. Such antibodies have, for example, beenproposed to target immune system cells to unwanted cells (U.S. Pat. No.4,676,980), and for treatment of HIV infection (WO 91/00360; WO92/200373; EP 03089). It is contemplated that the antibodies can beprepared in vitro using known methods in synthetic protein chemistry,including those involving crosslinking agents. For example, immunotoxinscan be constructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

[0199]8. Effector Function Engineering

[0200] It can be desirable to modify the FGF-CX antibody of theinvention with respect to effector function, so as to enhance, e.g., theeffectiveness of the antibody in treating cancer. For example, cysteineresidue(s) can be introduced into the Fc region, thereby allowinginterchain disulfide bond formation in this region. The homodimericantibody thus generated can have improved internalization capabilityand/or increased complement-mediated cell killing and antibody-dependentcellular cytotoxicity (ADCC). See Caron et al., J. Exp Med., 176:1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992).Homodimeric antibodies with enhanced anti-tumor activity can also beprepared using heterobifunctional cross-linkers as described in Wolff etal. Cancer Research, 53: 2560-2565 (1993). Alternatively, an antibodycan be engineered that has dual Fc regions and can thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al.,Anti-Cancer Drug Design, 3: 219-230 (1989).

[0201] 9. Immunoconjugates

[0202] The invention also pertains to immunoconjugates comprising aFGF-CX antibody conjugated to a cytotoxic agent such as achemotherapeutic agent, toxin (e.g., an enzymatically active toxin ofbacterial, fungal, plant, or animal origin, or fragments thereof), or aradioactive isotope (i.e., a radioconjugate).

[0203] Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated antibodies. Examples include 212Bi, 131I, 131In, 90Y,and 186Re.

[0204] Conjugates of the antibody and cytotoxic agent are made using avariety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1 -isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

[0205] In another embodiment, the antibody can be conjugated to a“receptor” (such streptavidin) for utilization in tumor pretargetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g., avidin) thatis in turn conjugated to a cytotoxic agent.

[0206] 10. Immunoliposomes

[0207] The antibodies disclosed herein can also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad.Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

[0208] Particularly useful liposomes can be generated by thereverse-phase evaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem., 257: 286-288 (1982) via a disulfide-interchange reaction. Achemotherapeutic agent (such as Doxorubicin) is optionally containedwithin the liposome. See Gabizon et al., J. National Cancer Inst.,81(19): 1484 (1989).

[0209] 11. Diagnostic Applications of Antibodies Directed Against theProteins of the Invention

[0210] Antibodies directed against a FGF-CX protein of the invention maybe used in methods known within the art relating to the localizationand/or quantitation of the protein (e.g., for use in measuring levels ofthe protein within appropriate physiological samples, for use indiagnostic methods, for use in imaging the protein, and the like). In agiven embodiment, antibodies against the proteins, or derivatives,fragments, analogs or homologs thereof, that contain the antigen bindingdomain, are utilized as pharmacologically-active compounds (see below).

[0211] An antibody specific for a FGF-CX protein of the invention can beused to isolate the protein by standard techniques, such asimmunoaffinity chromatography or immunoprecipitation. Such an antibodycan facilitate the purification of the natural protein antigen fromcells and of recombinantly produced antigen expressed in host cells.Moreover, such an antibody can be used to detect the antigenic protein(e.g., in a cellular lysate or cell supernatant) in order to evaluatethe abundance and pattern of expression of the antigenic protein.Antibodies directed against the FGF-CX protein can be useddiagnostically to monitor protein levels in tissue as part of a clinicaltesting procedure, e.g., to, for example, determine the efficacy of agiven treatment regimen. Detection can be facilitated by coupling (i.e.,physically linking) the antibody to a detectable substance. Examples ofdetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,and radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

[0212] 12. Antibody Therapeutics

[0213] FGF-CX antibodies of the invention, including polyclonal,monoclonal, humanized and fully human antibodies, may used astherapeutic agents. Such agents will generally be employed to treat orprevent a disease or pathology in a subject. An antibody preparation,preferably one having high specificity and high affinity for its targetantigen, is administered to the subject and will generally have aneffect due to its binding with the target. Such an effect may be one oftwo kinds, depending on the specific nature of the interaction betweenthe given antibody molecule and the target antigen in question. In thefirst instance, administration of the antibody may abrogate or inhibitthe binding of the target with an endogenous ligand to which itnaturally binds. In this case, the antibody binds to the target andmasks a binding site of the naturally occurring ligand, wherein theligand serves as an effector molecule. Thus the receptor mediates asignal transduction pathway for which ligand is responsible.

[0214] Alternatively, the effect may be one in which the antibodyelicits a physiological result by virtue of binding to an effectorbinding site on the target molecule. In this case the target, a receptorhaving an endogenous ligand which may be absent or defective in thedisease or pathology, binds the antibody as a surrogate effector ligand,initiating a receptor-based signal transduction event by the receptor.

[0215] A therapeutically effective amount of an antibody of theinvention relates generally to the amount needed to achieve atherapeutic objective. As noted above, this may be a binding interactionbetween the antibody and its target antigen that, in certain cases,interferes with the functioning of the target, and in other cases,promotes a physiological response. The amount required to beadministered will furthermore depend on the binding affinity of theantibody for its specific antigen, and will also depend on the rate atwhich an administered antibody is depleted from the free volume othersubject to which it is administered. Common ranges for therapeuticallyeffective dosing of an antibody or antibody fragment of the inventionmay be, by way of nonlimiting example, from about 0.1 mg/kg body weightto about 50 mg/kg body weight. Common dosing frequencies may range, forexample, from twice daily to once a week.

[0216] 13. Pharmaceutical Compositions of Antibodies

[0217] Antibodies specifically binding a FGF-CX protein of theinvention, as well as other molecules identified by the screening assaysdisclosed herein, can be administered for the treatment of variousdisorders in the form of pharmaceutical compositions. Principles andconsiderations involved in preparing such compositions, as well asguidance in the choice of components are provided, for example, inRemington: The Science And Practice Of Pharmacy 19th ed. (Alfonso R.Gennaro, et al., editors) Mack Pub. Co., Easton, Pa.: 1995; DrugAbsorption Enhancement: Concepts, Possibilities, Limitations, AndTrends, Harwood Academic Publishers, Langhorne, Pa., 1994; and PeptideAnd Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4),1991, M. Dekker, New York.

[0218] If the antigenic protein is intracellular and whole antibodiesare used as inhibitors, internalizing antibodies are preferred. However,liposomes can also be used to deliver the antibody, or an antibodyfragment, into cells. Where antibody fragments are used, the smallestinhibitory fragment that specifically binds to the binding domain of thetarget protein is preferred. For example, based upon the variable-regionsequences of an antibody, peptide molecules can be designed that retainthe ability to bind the target protein sequence. Such peptides can besynthesized chemically and/or produced by recombinant DNA technology.See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893(1993). The formulation herein can also contain more than one activecompound as necessary for the particular indication being treated,preferably those with complementary activities that do not adverselyaffect each other. Alternatively, or in addition, the composition cancomprise an agent that enhances its function, such as, for example, acytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitoryagent. Such molecules are suitably present in combination in amountsthat are effective for the purpose intended.

[0219] The active ingredients can also be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacrylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions.

[0220] The formulations to be used for in vivo administration must besterile. This is readily accomplished by filtration through sterilefiltration membranes.

[0221] FGF-CX Recombinant Expression Vectors and Host Cells

[0222] Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding FGF-CX protein,or derivatives, fragments, analogs or homologs thereof. As used herein,the term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid”, which refers to a circular double stranded DNAloop into which additional DNA segments can be ligated. Another type ofvector is a viral vector, wherein additional DNA segments can be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)are integrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “expression vectors”. In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids.In the present specification, “plasmid” and “vector” can be usedinterchangeably as the plasmid is the most commonly used form of vector.However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions.

[0223] The recombinant expression vectors of the invention comprise anucleic acid of the invention in a form suitable for expression of thenucleic acid in a host cell, which means that the recombinant expressionvectors include one or more regulatory sequences, selected on the basisof the host cells to be used for expression, that is operatively linkedto the nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory sequence(s)in a manner that allows for expression of the nucleotide sequence (e.g.,in an in vitro transcription/translation system or in a host cell whenthe vector is introduced into the host cell). The term “regulatorysequence” is intended to includes promoters, enhancers and otherexpression control elements (e.g., polyadenylation signals). Suchregulatory sequences are described, for example, in Goeddel; GENEEXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, SanDiego, Calif. (1990). Regulatory sequences include those that directconstitutive expression of a nucleotide sequence in many types of hostcell and those that direct expression of the nucleotide sequence only incertain host cells (e.g., tissue-specific regulatory sequences). It willbe appreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression of protein desired, etc.The expression vectors of the invention can be introduced into hostcells to thereby produce proteins or peptides, including fusion proteinsor peptides, encoded by nucleic acids as described herein (e.g., FGF-CXproteins, mutant forms of FGF-CX, fusion proteins, etc.).

[0224] The recombinant expression vectors of the invention can bedesigned for expression of FGF-CX in prokaryotic or eukaryotic cells.For example, FGF-CX can be expressed in bacterial cells such as E. coli,insect cells (using baculovirus expression vectors) yeast cells ormammalian cells. Suitable host cells are discussed further in Goeddel,GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press,San Diego, Calif. (1990). Alternatively, the recombinant expressionvector can be transcribed and translated in vitro, for example using T7promoter regulatory sequences and T7 polymerase.

[0225] Expression of proteins in prokaryotes is most often carried outin E. coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: (1) to increase expression ofrecombinant protein; (2) to increase the solubility of the recombinantprotein; and (3) to aid in the purification of the recombinant proteinby acting as a ligand in affinity purification. Often, in fusionexpression vectors, a proteolytic cleavage site is introduced at thejunction of the fusion moiety and the recombinant protein to enableseparation of the recombinant protein from the fusion moiety subsequentto purification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith and Johnson (1988) Gene 67:31-40), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein.

[0226] Examples of suitable inducible non-fusion E. coli expressionvectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d(Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185,Academic Press, San Diego, Calif. (1990) 60-89).

[0227] One strategy to maximize recombinant protein expression in E.coli is to express the protein in a host bacteria with an impairedcapacity to proteolytically cleave the recombinant protein. See,Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185,Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is toalter the nucleic acid sequence of the nucleic acid to be inserted intoan expression vector so that the individual codons for each amino acidare those preferentially utilized in E. coli (Wada et al., (1992)Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acidsequences of the invention can be carried out by standard DNA synthesistechniques.

[0228] In another embodiment, the FGF-CX expression vector is a yeastexpression vector. Examples of vectors for expression in yeast S.cerivisae include pYepSec1 (Baldari, et al., (1987) EMBO J 6:229-234),pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz etal., (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego,Calif.), and picZ (In Vitrogen Corp, San Diego, Calif.).

[0229] Alternatively, FGF-CX can be expressed in insect cells usingbaculovirus expression vectors. Baculovirus vectors available forexpression of proteins in cultured insect cells (e.g., SF9 cells)include the pAc series (Smith et al. (1983) Mol Cell Biol 3:2156-2165)and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).

[0230] In yet another embodiment, a nucleic acid of the invention isexpressed in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed (1987)Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J 6: 187-195).When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, Adenovirus 2, cytomegalovirusand Simian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells. See, e.g., Chapters 16 and 17 ofSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

[0231] In another embodiment, the recombinant mammalian expressionvector is capable of directing expression of the nucleic acidpreferentially in a particular cell type (e.g., tissue-specificregulatory elements are used to express the nucleic acid).Tissue-specific regulatory elements are known in the art. Non-limitingexamples of suitable tissue-specific promoters include the albuminpromoter (liver-specific; Pinkert et al. (1987) Genes Dev 1:268-277),lymphoid-specific promoters (Calame and Eaton (1988) Adv Immunol43:235-275), in particular promoters of T cell receptors (Winoto andBaltimore (1989) EMBO J 8:729-733) and immunoglobulins (Banerji et al.(1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748),neuron-specific promoters (e.g., the neurofilament promoter; Byrne andRuddle (1989) PNAS 86:5473-5477), pancreas-specific promoters (Edlund etal. (1985) Science 230:912-916), and mammary gland-specific promoters(e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and EuropeanApplication Publication No. 264,166). Developmentally-regulatedpromoters are also encompassed, e.g., the murine hox promoters (Kesseland Gruss (1990) Science 249:374-379) and the α-fetoprotein promoter(Campes and Tilghman (1989) Genes Dev 3:537-546).

[0232] The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner that allows forexpression (by transcription of the DNA molecule) of an RNA moleculethat is antisense to FGF-CX mRNA. Regulatory sequences operativelylinked to a nucleic acid cloned in the antisense orientation can bechosen that direct the continuous expression of the antisense RNAmolecule in a variety of cell types, for instance viral promoters and/orenhancers, or regulatory sequences can be chosen that directconstitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes see Weintraub etal., “Antisense RNA as a molecular tool for genetic analysis,”Reviews--Trends in Genetics, Vol. 1(1) 1986.

[0233] Another aspect of the invention pertains to host cells into whicha recombinant expression vector of the invention has been introduced.The terms “host cell” and “recombinant host cell” are usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

[0234] A host cell can be any prokaryotic or eukaryotic cell. Forexample, FGF-CX protein can be expressed in bacterial cells such as E.coli, insect cells, yeast or mammalian cells (such as Chinese hamsterovary cells (CHO) or COS cells). Other suitable host cells are known tothose skilled in the art.

[0235] Vector DNA can be introduced into prokaryotic or eukaryotic cellsvia conventional transformation or transfection techniques. As usedherein, the terms “transformation” and “transfection” are intended torefer to a variety of art-recognized techniques for introducing foreignnucleic acid (e.g., DNA) into a host cell, including calcium phosphateor calcium chloride co-precipitation, DEAE-dextran-mediatedtransfection, lipofection, or electroporation. Suitable methods fortransforming or transfecting host cells can be found in Sambrook, et a.(MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989), and other laboratory manuals.

[0236] For stable transfection of mammalian cells, it is known that,depending upon the expression vector and transfection technique used,only a small fraction of cells may integrate the foreign DNA into theirgenome. In order to identify and select these integrants, a gene thatencodes a selectable marker (e.g., resistance to antibiotics) isgenerally introduced into the host cells along with the gene ofinterest. Various selectable markers include those that conferresistance to drugs, such as G418, hygromycin and methotrexate. Nucleicacid encoding a selectable marker can be introduced into a host cell onthe same vector as that encoding FGF-CX or can be introduced on aseparate vector. Cells stably transfected with the introduced nucleicacid can be identified by drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

[0237] A host cell of the invention, such as a prokaryotic or eukaryotichost cell in culture, can be used to produce (i.e., express) FGF-CXprotein. Accordingly, the invention further provides methods forproducing FGF-CX protein using the host cells of the invention. In oneembodiment, the method comprises culturing the host cell of invention(into which a recombinant expression vector encoding FGF-CX has beenintroduced) in a suitable medium such that FGF-CX protein is produced.In another embodiment, the method further comprises isolating FGF-CXfrom the medium or the host cell.

[0238] Transgenic Animals

[0239] The host cells of the invention can also be used to producenonhuman transgenic animals. For example, in one embodiment, a host cellof the invention is a fertilized oocyte or an embryonic stem cell intowhich FGF-CX-coding sequences have been introduced. Such host cells canthen be used to create non-human transgenic animals in which exogenousFGF-CX sequences have been introduced into their genome or homologousrecombinant animals in which endogenous FGF-CX sequences have beenaltered. Such animals are useful for studying the function and/oractivity of FGF-CX and for identifying and/or evaluating modulators ofFGF-CX activity. As used herein, a “transgenic animal” is a non-humananimal, preferably a mammal, more preferably a rodent such as a rat ormouse, in which one or more of the cells of the animal includes atransgene. Other examples of transgenic animals include non-humanprimates, sheep, dogs, cows, goats, chickens, amphibians, etc. Atransgene is exogenous DNA that is integrated into the genome of a cellfrom which a transgenic animal develops and that remains in the genomeof the mature animal, thereby directing the expression of an encodedgene product in one or more cell types or tissues of the transgenicanimal. As used herein, a “homologous recombinant animal” is a non-humananimal, preferably a mammal, more preferably a mouse, in which anendogenous FGF-CX gene has been altered by homologous recombinationbetween the endogenous gene and an exogenous DNA molecule introducedinto a cell of the animal, e.g., an embryonic cell of the animal, priorto development of the animal.

[0240] A transgenic animal of the invention can be created byintroducing FGF-CX-encoding nucleic acid into the male pronuclei of afertilized oocyte, e.g., by microinjection, retroviral infection, andallowing the oocyte to develop in a pseudopregnant female foster animal.The human FGF-CX DNA sequence of SEQ ID NO: 1 can be introduced as atransgene into the genome of a non-human animal. Alternatively, anonhuman homologue of the human FGF-CX gene, such as a mouse FGF-CXgene, can be isolated based on hybridization to the human FGF-CX cDNA(described further above) and used as a transgene. Intronic sequencesand polyadenylation signals can also be included in the transgene toincrease the efficiency of expression of the transgene. Atissue-specific regulatory sequence(s) can be operably linked to theFGF-CX transgene to direct expression of FGF-CX protein to particularcells. Methods for generating transgenic animals via embryo manipulationand microinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866; 4,870,009; and 4,873,191; and Hogan 1986, In:MANIPULATING THE MOUSE EMBRYO, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. Similar methods are used for production of othertransgenic animals. A transgenic founder animal can be identified basedupon the presence of the FGF-CX transgene in its genome and/orexpression of FGF-CX mRNA in tissues or cells of the animals. Atransgenic founder animal can then be used to breed additional animalscarrying the transgene. Moreover, transgenic animals carrying atransgene encoding FGF-CX can further be bred to other transgenicanimals carrying other transgenes.

[0241] To create a homologous recombinant animal, a vector is preparedwhich contains at least a portion of a FGF-CX gene into which adeletion, addition or substitution has been introduced to thereby alter,e.g., functionally disrupt, the FGF-CX gene. The FGF-CX gene can be ahuman gene (e.g., SEQ ID NO: 1), but more preferably, is a non-humanhomologue of a human FGF-CX gene. For example, a mouse homologue ofhuman FGF-CX gene of SEQ ID NO: 1 can be used to construct a homologousrecombination vector suitable for altering an endogenous FGF-CX gene inthe mouse genome. In one embodiment, the vector is designed such that,upon homologous recombination, the endogenous FGF-CX gene isfunctionally disrupted (i.e., no longer encodes a functional protein;also referred to as a “knock out” vector).

[0242] Alternatively, the vector can be designed such that, uponhomologous recombination, the endogenous FGF-CX gene is mutated orotherwise altered but still encodes functional protein (e.g., theupstream regulatory region can be altered to thereby alter theexpression of the endogenous FGF-CX protein). In the homologousrecombination vector, the altered portion of the FGF-CX gene is flankedat its 5′ and 3′ ends by additional nucleic acid of the FGF-CX gene toallow for homologous recombination to occur between the exogenous FGF-CXgene carried by the vector and an endogenous FGF-CX gene in an embryonicstem cell. The additional flanking FGF-CX nucleic acid is of sufficientlength for successful homologous recombination with the endogenous gene.Typically, several kilobases of flanking DNA (both at the 5′ and 3′ends) are included in the vector. See e.g., Thomas et al. (1987) Cell51:503 for a description of homologous recombination vectors. The vectoris introduced into an embryonic stem cell line (e.g., byelectroporation) and cells in which the introduced FGF-CX gene hashomologously recombined with the endogenous FGF-CX gene are selected(see e.g., Li et al. (1992) Cell 69:915).

[0243] The selected cells are then injected into a blastocyst of ananimal (e.g., a mouse) to 25 form aggregation chimeras. See e.g.,Bradley 1987, In: TERATOCARCINOMAS AND EMBRYONIC STEM CELLS: A PRACTICALAPPROACH, Robertson, ed. IRL, Oxford, pp. 113-152. A chimeric embryo canthen be implanted into a suitable pseudopregnant female foster animaland the embryo brought to term. Progeny harboring the homologouslyrecombined DNA in their germ cells can be used to breed animals in whichall cells of the animal contain the homologously recombined DNA bygermline transmission of the transgene. Methods for constructinghomologous recombination vectors and homologous recombinant animals aredescribed further in Bradley (1991) Curr Opin Biotechnol 2:823-829; PCTInternational Publication Nos.: WO 90/11354; WO 91/01140; WO 92/0968;and WO 93/04169.

[0244] In another embodiment, transgenic non-humans animals can beproduced that contain selected systems that allow for regulatedexpression of the transgene. One example of such a system is thecre/loxP recombinase system of bacteriophage P1. For a description ofthe cre/loxP recombinase system, see, e.g., Lakso et al. (1992) PNAS89:6232-6236. Another example of a recombinase system is the FLPrecombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991)Science 251:1351-1355. If a cre/loxP recombinase system is used toregulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein are required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

[0245] Clones of the non-human transgenic animals described herein canalso be produced according to the methods described in Wilmut et al.(1997) Nature 385:810-813. In brief, a cell, e.g., a somatic cell, fromthe transgenic animal can be isolated and induced to exit the growthcycle and enter G₀ phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyte and then transferred to pseudopregnant femalefoster animal. The offspring borne of this female foster animal will bea clone of the animal from which the cell, e.g., the somatic cell, isisolated.

[0246] Pharmaceutical Compositions

[0247] The FGF-CX nucleic acid molecules, FGF-CX proteins, andanti-FGF-CX antibodies (also referred to herein as “active compounds”)of the invention, and derivatives, fragments, analogs and homologsthereof, can be incorporated into pharmaceutical compositions suitablefor administration. Such compositions typically comprise the nucleicacid molecule, protein, or antibody and a pharmaceutically acceptablecarrier. As used herein, “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration.Suitable carriers are described in the most recent edition ofRemington's Pharmaceutical Sciences, a standard reference text in thefield, which is incorporated herein by reference. Preferred examples ofsuch carriers or diluents include, but are not limited to, water,saline, Ringer's solutions, dextrose solution, and 5% human serumalbumin. Liposomes and non-aqueous vehicles such as fixed oils may alsobe used. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active compound, use thereof inthe compositions is contemplated. Supplementary active compounds canalso be incorporated into the compositions.

[0248] A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates, and agents for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

[0249] Pharmaceutical compositions suitable for injectable use includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

[0250] Sterile injectable solutions can be prepared by incorporating theactive compound (e.g., a FGF-CX protein or anti-FGF-CX antibody) in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, methods of preparation are vacuum drying and freeze-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

[0251] Oral compositions generally include an inert diluent or an ediblecarrier. They can be o enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

[0252] For administration by inhalation, the compounds are delivered inthe form of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

[0253] Systemic administration can also be by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration, detergents, bilesalts, and fusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

[0254] The compounds can also be prepared in the form of suppositories(e.g., with conventional suppository bases such as cocoa butter andother glycerides) or retention enemas for rectal delivery.

[0255] In one embodiment, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

[0256] It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved.

[0257] The nucleic acid molecules of the invention can be inserted intovectors and used as gene therapy vectors. Gene therapy vectors can bedelivered to a subject by any of a number of routes, e.g., as describedin U.S. Pat. Nos. 5,703,055. Delivery can thus also include, e.g.,intravenous injection, local administration (see U.S. Pat. No.5,328,470) or stereotactic injection (see e.g., Chen et al. (1994) PNAS91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells that producethe gene delivery system.

[0258] The pharmaceutical compositions can be included in a container,pack, or dispenser together with instructions for administration.

[0259] Uses and Methods of the Invention

[0260] The nucleic acid molecules, proteins, protein homologues, andantibodies described herein can be used in one or more of the followingmethods: (a) screening assays; (b) detection assays (e.g., chromosomalmapping, tissue typing, forensic biology), (c) predictive medicine(e.g., diagnostic assays, prognostic assays, monitoring clinical trials,and pharmacogenomics); and (d) methods of treatment (e.g., therapeuticand prophylactic). As described herein, in one embodiment, a FGF-CXprotein of the invention has the ability to bind ATP.

[0261] The isolated nucleic acid molecules of the invention can be usedto express FGF-CX protein (e.g., via a recombinant expression vector ina host cell in gene therapy applications), to detect FGF-CX mRNA (e.g.,in a biological sample) or a genetic lesion in a FGF-CX gene, and tomodulate FGF-CX activity, as described further below. In addition, theFGF-CX proteins can be used to screen drugs or compounds that modulatethe FGF-CX activity or expression as well as to treat disorderscharacterized by insufficient or excessive production of FGF-CX protein,for example proliferative or differentiative disorders, or production ofFGF-CX protein forms that have decreased or aberrant activity comparedto FGF-CX wild type protein. In addition, the anti-FGF-CX antibodies ofthe invention can be used to detect and isolate FGF-CX proteins andmodulate FGF-CX activity.

[0262] This invention further pertains to novel agents identified by theabove described screening assays and uses thereof for treatments asdescribed herein.

[0263] Screening Assays

[0264] The invention provides a method (also referred to herein as a“screening assay”) for identifying modulators, i.e., candidate or testcompounds or agents (e.g. peptides, peptidomimetics, small molecules orother drugs) that bind to FGF-CX proteins or have a stimulatory orinhibitory effect on, for example, FGF-CX expression or FGF-CX activity.

[0265] In one embodiment, the invention provides assays for screeningcandidate or test compounds which bind to or modulate the activity of aFGF-CX protein or polypeptide or biologically active portion thereof.The test compounds of the present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart, including: biological libraries; spatially addressable parallelsolid phase or solution phase libraries; synthetic library methodsrequiring deconvolution; the “one-bead one-compound” library method; andsynthetic library methods using affinity chromatography selection. Thebiological library approach is limited to peptide libraries, while theother four approaches are applicable to peptide, non-peptide oligomer orsmall molecule libraries of compounds (Lam (1997) Anticancer Drug Des12:145).

[0266] Examples of methods for the synthesis of molecular libraries canbe found in the art, for example in: DeWitt et al. (1993) Proc Natl AcadSci U.S.A. 90:6909; Erb et al. (1994) Proc Natl Acad Sci U.S.A.91:11422; Zuckermann et al. (1994) J Med Chem 37:2678; Cho et al. (1993)Science 261:1303; Carrell et al. (1994) Angew Chem Int Ed Engl 33:2059;Carell et al. (1994) Angew Chem Int Ed Engl 33:2061; and Gallop et al.(1994) J Med Chem 37:1233.

[0267] Libraries of compounds may be presented in solution (e.g.,Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991)Nature 354:82-84), on chips (Fodor (1993) Nature 364:555-556), bacteria(Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409),plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or onphage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science249:404-406; Cwirla et al. (1990) Proc Natl Acad Sci U.S.A.87:6378-6382; Felici (1991) J Mol Biol 222:301-310; Ladner above.).

[0268] In one embodiment, an assay is a cell-based assay in which a cellwhich expresses a membrane-bound form of FGF-CX protein, or abiologically active portion thereof, on the cell surface is contactedwith a test compound and the ability of the test compound to bind to aFGF-CX protein determined. The cell, for example, can of mammalianorigin or a yeast cell. Determining the ability of the test compound tobind to the FGF-CX protein can be accomplished, for example, by couplingthe test compound with a radioisotope or enzymatic label such thatbinding of the test compound to the FGF-CX protein or biologicallyactive portion thereof can be determined by detecting the labeledcompound in a complex. For example, test compounds can be labeled with¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, and theradioisotope detected by direct counting of radioemission or byscintillation counting. Alternatively, test compounds can beenzymatically labeled with, for example, horseradish peroxidase,alkaline phosphatase, or luciferase, and the enzymatic label detected bydetermination of conversion of an appropriate substrate to product. Inone embodiment, the assay comprises contacting a cell which expresses amembrane-bound form of FGF-CX protein, or a biologically active portionthereof, on the cell surface with a known compound which binds FGF-CX toform an assay mixture, contacting the assay mixture with a testcompound, and determining the ability of the test compound to interactwith a FGF-CX protein, wherein determining the ability of the testcompound to interact with a FGF-CX protein comprises determining theability of the test compound to preferentially bind to FGF-CX or abiologically active portion thereof as compared to the known compound.

[0269] In another embodiment, an assay is a cell-based assay comprisingcontacting a cell expressing a membrane-bound form of FGF-CX protein, ora biologically active portion thereof, on the cell surface with a testcompound and determining the ability of the test compound to modulate(e.g., stimulate or inhibit) the activity of the FGF-CX protein orbiologically active portion thereof. Determining the ability of the testcompound to modulate the activity of FGF-CX or a biologically activeportion thereof can be accomplished, for example, by determining theability of the FGF-CX protein to bind to or interact with a FGF-CXtarget molecule. As used herein, a “target molecule” is a molecule withwhich a FGF-CX protein binds or interacts in nature, for example, amolecule on the surface of a cell which expresses a FGF-CX interactingprotein, a molecule on the surface of a second cell, a molecule in theextracellular milieu, a molecule associated with the internal surface ofa cell membrane or a cytoplasmic molecule. A FGF-CX target molecule canbe a non-FGF-CX molecule or a FGF-CX protein or polypeptide of thepresent invention. In one embodiment, a FGF-CX target molecule is acomponent of a signal transduction pathway that facilitates transductionof an extracellular signal (e.g., a signal generated by binding of acompound to a membrane-bound FGF-CX molecule) through the cell membraneand into the cell. The target, for example, can be a secondintercellular protein that has catalytic activity or a protein thatfacilitates the association of downstream signaling molecules withFGF-CX.

[0270] Determining the ability of the FGF-CX protein to bind to orinteract with a FGF-CX target molecule can be accomplished by one of themethods described above for determining direct binding. In oneembodiment, determining the ability of the FGF-CX protein to bind to orinteract with a FGF-CX target molecule can be accomplished bydetermining the activity of the target molecule. For example, theactivity of the target molecule can be determined by detecting inductionof a cellular second messenger of the target (i.e. intracellular Ca²⁺,diacylglycerol, IP₃, etc.), detecting catalytic/enzymatic activity ofthe target an appropriate substrate, detecting the induction of areporter gene (comprising a FGF-CX-responsive regulatory elementoperatively linked to a nucleic acid encoding a detectable marker, e.g.,luciferase), or detecting a cellular response, for example, cellsurvival, cellular differentiation, or cell proliferation.

[0271] In yet another embodiment, an assay of the present invention is acell-free assay comprising contacting a FGF-CX protein or biologicallyactive portion thereof with a test compound and determining the abilityof the test compound to bind to the FGF-CX protein or biologicallyactive portion thereof. Binding of the test compound to the FGF-CXprotein can be determined either directly or indirectly as describedabove. In one embodiment, the assay comprises contacting the FGF-CXprotein or biologically active portion thereof with a known compoundwhich binds FGF-CX to form an assay mixture, contacting the assaymixture with a test compound, and determining the ability of the testcompound to interact with a FGF-CX protein, wherein determining theability of the test compound to interact with a FGF-CX protein comprisesdetermining the ability of the test compound to preferentially bind toFGF-CX or biologically active portion thereof as compared to the knowncompound.

[0272] In another embodiment, an assay is a cell-free assay comprisingcontacting FGF-CX protein or biologically active portion thereof with atest compound and determining the ability of the test compound tomodulate (e.g., stimulate or inhibit) the activity of the FGF-CX proteinor biologically active portion thereof. Determining the ability of thetest compound to modulate the activity of FGF-CX can be accomplished,for example, by determining the ability of the FGF-CX protein to bind toa FGF-CX target molecule by one of the methods described above fordetermining direct binding. In an alternative embodiment, determiningthe ability of the test compound to modulate the activity of FGF-CX canbe accomplished by determining the ability of the FGF-CX protein furthermodulate a FGF-CX target molecule. For example, the catalytic/enzymaticactivity of the target molecule on an appropriate substrate can bedetermined as previously described.

[0273] In yet another embodiment, the cell-free assay comprisescontacting the FGF-CX protein or biologically active portion thereofwith a known compound which binds FGF-CX to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to interact with a FGF-CX protein, whereindetermining the ability of the test compound to interact with a FGF-CXprotein comprises determining the ability of the FGF-CX protein topreferentially bind to or modulate the activity of a FGF-CX targetmolecule.

[0274] The cell-free assays of the present invention are amenable to useof both the soluble form or the membrane-bound form of FGF-CX. In thecase of cell-free assays comprising the membrane-bound form of FGF-CX,it may be desirable to utilize a solubilizing agent such that themembrane-bound form of FGF-CX is maintained in solution. Examples ofsuch solubilizing agents include non-ionic detergents such asn-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside,octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100,Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)_(n) ,N-dodecyl--N,N-dimethyl-3-ammonio-1-propane sulfonate,3-(3-cholamidopropyl)dimethylamminiol-1-propane sulfonate (CHAPS), or3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate(CHAPSO).

[0275] In more than one embodiment of the above assay methods of thepresent invention, it may be desirable to immobilize either FGF-CX orits target molecule to facilitate separation of complexed fromuncomplexed forms of one or both of the proteins, as well as toaccommodate automation of the assay. Binding of a test compound toFGF-CX, or interaction of FGF-CX with a target molecule in the presenceand absence of a candidate compound, can be accomplished in any vesselsuitable for containing the reactants. Examples of such vessels includemicrotiter plates, test tubes, and micro-centrifuge tubes. In oneembodiment, a fusion protein can be provided that adds a domain thatallows one or both of the proteins to be bound to a matrix. For example,GST-FGF-CX fusion proteins or GST-target fusion proteins can be adsorbedonto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtiter plates, that are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or FGF-CX protein, and the mixture is incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotiter plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, complex determined eitherdirectly or indirectly, for example, as described above. Alternatively,the complexes can be dissociated from the matrix, and the level ofFGF-CX binding or activity determined using standard techniques.

[0276] Other techniques for immobilizing proteins on matrices can alsobe used in the screening assays of the invention. For example, eitherFGF-CX or its target molecule can be immobilized utilizing conjugationof biotin and streptavidin. Biotinylated FGF-CX or target molecules canbe prepared from biotin-NHS (N-hydroxy-succinimide) using techniqueswell known in the art (e.g., biotinylation kit, Pierce Chemicals,Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96well plates (Pierce Chemical). Alternatively, antibodies reactive withFGF-CX or target molecules, but which do not interfere with binding ofthe FGF-CX protein to its target molecule, can be derivatized to thewells of the plate, and unbound target or FGF-CX trapped in the wells byantibody conjugation. Methods for detecting such complexes, in additionto those described above for the GST-immobilized complexes, includeimmunodetection of complexes using antibodies reactive with the FGF-CXor target molecule, as well as enzyme-linked assays that rely ondetecting an enzymatic activity associated with the FGF-CX or targetmolecule.

[0277] In another embodiment, modulators of FGF-CX expression areidentified in a method wherein a cell is contacted with a candidatecompound and the expression of FGF-CX mRNA or protein in the cell isdetermined. The level of expression of FGF-CX mRNA or protein in thepresence of the candidate compound is compared to the level ofexpression of FGF-CX mRNA or protein in the absence of the candidatecompound. The candidate compound can then be identified as a modulatorof FGF-CX expression based on this comparison. For example, whenexpression of FGF-CX mRNA or protein is greater (statisticallysignificantly greater) in the presence of the candidate compound than inits absence, the candidate compound is identified as a stimulator ofFGF-CX mRNA or protein expression. Alternatively, when expression ofFGF-CX mRNA or protein is less (statistically significantly less) in thepresence of the candidate compound than in its absence, the candidatecompound is identified as an inhibitor of FGF-CX mRNA or proteinexpression. The level of FGF-CX mRNA or protein expression in the cellscan be determined by methods described herein for detecting FGF-CX mRNAor protein.

[0278] In yet another aspect of the invention, the FGF-CX proteins canbe used as “bait in we proteins” in a two-hybrid assay or three hybridassay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J Biol Chem 268:12046-12054; Bartel etal. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent W094/10300), to identify other proteins that bindto or interact with FGF-CX (“FGF-CX-binding proteins” or “FGF-CX-bp”)and modulate FGF-CX activity. Such FGF-CX-binding proteins are alsolikely to be involved in the propagation of signals by the FGF-CXproteins as, for example, upstream or downstream elements of the FGF-CXpathway.

[0279] The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for FGF-CX is fused toa gene encoding the DNA binding domain of a known transcription factor(e g., GAL-4). In the other construct, a DNA sequence, from a library ofDNA sequences, that encodes an unidentified protein (“prey” or “sample”)is fused to a gene that codes for the activation domain of the knowntranscription factor. If the “bait” and the “prey” proteins are able tointeract, in vivo, forming a FGF-CX-dependent complex, the DNA-bindingand activation domains of the transcription factor are brought intoclose proximity. This proximity allows transcription of a reporter gene(e.g., LacZ) that is operably linked to a transcriptional regulatorysite responsive to the transcription factor. Expression of the reportergene can be detected and cell colonies containing the functionaltranscription factor can be isolated and used to obtain the cloned genethat encodes the protein which interacts with FGF-CX.

[0280] This invention further pertains to novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

[0281] Detection Assays

[0282] Portions or fragments of the cDNA sequences identified herein(and the corresponding complete gene sequences) can be used in numerousways as polynucleotide reagents. For example, these sequences can beused to: (i) map their respective genes on a chromosome; and, thus,locate gene regions associated with genetic disease; (ii) identify anindividual from a minute biological sample (tissue typing); and (iii)aid in forensic identification of a biological sample.

[0283] The FGF-CX sequences of the present invention can also be used toidentify individuals from minute biological samples. In this technique,an individual's genomic DNA is digested with one or more restrictionenzymes, and probed on a Southern blot to yield unique bands foridentification. The sequences of the present invention are useful asadditional DNA markers for RFLP (“restriction fragment lengthpolymorphisms,” described in U.S. Pat. No. 5,272,057).

[0284] Furthermore, the sequences of the present invention can be usedto provide an alternative technique that determines the actualbase-by-base DNA sequence of selected portions of an individual'sgenome. Thus, the FGF-CX sequences described herein can be used toprepare two PCR primers from the 5′ and 3′ ends of the sequences. Theseprimers can then be used to amplify an individual's DNA and subsequentlysequence it.

[0285] Panels of corresponding DNA sequences from individuals, preparedin this manner, can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences due to allelicdifferences. The sequences of the present invention can be used toobtain such identification sequences from individuals and from tissue.The FGF-CX sequences of the invention uniquely represent portions of thehuman genome. Allelic variation occurs to some degree in the codingregions of these sequences, and to a greater degree in the noncodingregions. It is estimated that allelic variation between individualhumans occurs with a frequency of about once per each 500 bases. Much ofthe allelic variation is due to single nucleotide polymorphisms (SNPs),which include restriction fragment length polymorphisms (RFLPs).

[0286] Each of the sequences described herein can, to some degree, beused as a standard against which DNA from an individual can be comparedfor identification purposes. Because greater numbers of polymorphismsoccur in the noncoding regions, fewer sequences are necessary todifferentiate individuals. The noncoding sequences of SEQ ID NO: 1, asdescribed above, can comfortably provide positive individualidentification with a panel of perhaps 10 to 1,000 primers that eachyield a noncoding amplified sequence of 100 bases. If predicted codingsequences are used, a more appropriate number of primers for positiveindividual identification would be 500-2,000.

[0287] Predictive Medicine

[0288] The present invention also pertains to the field of predictivemedicine in which diagnostic assays, prognostic assays,pharmacogenomics, and monitoring clinical trials are used for prognostic(predictive) purposes to thereby treat an individual prophylactically.Accordingly, one aspect of the present invention relates to diagnosticassays for determining FGF-CX protein and/or nucleic acid expression aswell as FGF-CX activity, in the context of a biological sample (e.g.,blood, serum, cells, tissue) to thereby determine whether an individualis afflicted with a disease or disorder, or is at risk of developing adisorder, associated with aberrant FGF-CX expression or activity. Theinvention also provides for prognostic (or predictive) assays fordetermining whether an individual is at risk of developing a disorderassociated with FGF-CX protein, nucleic acid expression or activity. Forexample, mutations in a FGF-CX gene can be assayed in a biologicalsample. Such assays can be used for prognostic or predictive purpose tothereby prophylactically treat an individual prior to the onset of adisorder characterized by or associated with FGF-CX protein, nucleicacid expression or activity.

[0289] Another aspect of the invention provides methods for determiningFGF-CX protein, nucleic acid expression or FGF-CX activity in anindividual to thereby select appropriate therapeutic or prophylacticagents for that individual (referred to herein as “pharmacogenomics”).Pharmacogenomics allows for the selection of agents (e.g., drugs) fortherapeutic or prophylactic treatment of an individual based on thegenotype of the individual (e.g., the genotype of the individualexamined to determine the ability of the individual to respond to aparticular agent.) Yet another aspect of the invention pertains tomonitoring the influence of agents (e.g., drugs, compounds) on theexpression or activity of FGF-CX in clinical trials.

[0290] These and other agents are described in further detail in thefollowing sections.

[0291] Diagnostic Assays

[0292] Fibroblast growth factors FGF-1 through FGF-9 generally promotecell proliferation in cells carrying the particular growth factorreceptor. Examples of FGF growth promotion include epithelial cells,such as fibroblasts and keratinocytes, in the anterior eye aftersurgery. Other conditions in which proliferation of cells plays a roleinclude tumors, restenosis, psoriasis, Dupuytren's contracture, diabeticcomplications, Kaposi's sarcoma and rheumatoid arthritis.

[0293] FGF-CX may be used in the method of the invention for detectingits corresponding fibroblast growth factor receptor CX (FGFRCX) in asample or tissue. The method comprises contacting the sample or tissuewith FGF-CX, allowing formation of receptor-ligand pairs, and detectingany FGFRCX: FGF-CX pairs. Compositions containing FGF-CX can be used toincrease FGFRCX activity, for example to stimulate cartilage or bonerepair. Compositions containing FGF-CX antagonists or FGF-CX bindingagents (e.g. anti- FGF-CX antibodies) can be used to treat diseasescaused by an excess of FGF-CX or overactivity of FGFRCX, especiallymultiple or solitary hereditary exostosis, hallux valgus deformity,achondroplasia, synovial chondromatosis and endochondromas.

[0294] Glia activating factor (GAF) and the DNA encoding GAF act tospecifically promote growth of glial cells. Some examples ofglia-associated disorders in which GAF may be utilized to modulate glialcell activities are cerebral lesions, cerebral edema, senile dementia,Alzheimer's disease, diabetic neuropathies, etc. Similarly, FGF-CX maybe used in diagnosis or treating glial cell related disorders. Theglial-cell modulating activity of FGF-CX may be as aneuroprotective-like activity, and FGF-CX may be used as aneuroprotective agent. Due to the close homology of FGF-CX to FGF-9,which was identified originally as a glia activating factor, it can bepresumed that the FGF-CX sequence is also a glia activating factor.FGF-CX can therefor be used to stimulate the growth of glia cells andcan be used to accelerate healing of cerebral lesions or to treatcerebral edema, senile dementia, Alzheimer's disease, or diabeticneuropathy.

[0295] FGF-CX can also be used to stimulates fibroblasts (foraccelerating healing of bums, wounds, ulcers, etc), megakaryocytes (toincrease the number of platelets), hematopoietic cells, immune systemcells, and vascular smooth muscle cells. FGF-CX is also expected to haveosteogenesis-promoting activity, and can be used for treating bonefractures and osteoporosis. Assay of FGF-CX polypeptide or nucleic acidmoieties may be useful in diagnosis of cerebral tumors, and antibodiesagainst could be used to treat such tumors. It can also be used as areagent for stimulating growth of cultured cells. An anticipated dosageis 1 ng-0.1 mg/kg/day, though treatment may vary depending on the typeor severity of the disorder being treated. FGF-CX polypeptides may beused as platelet increasing agents, osteogenesis promoting agents or fortreating cerebral nervous diseases or hepatopathy such as hepaticcirrhosis. They can also be used to treat cancer when used alongside ananticancer agent. Antibodies directed against the FGF-CX polypeptide, orfragments, derivatives, or analogs thereof, can be used for detecting ordetermining a biological activity of a FGF-CX polypeptide or forpurifying a FGF-CX polypeptide. Those antibodies that also neutralizethe cell growth activity of FGF-CX can be used as anticancer agents.

[0296] Many, if not all, homologous proteins are known in the art tohave closely related or identical functions. See, e.g., Lewin, “Chapter21: Structural Genes Belong to Families” In: GENES II, 1985, John Wileyand Sons, Inc., New York. The FGF-CX polypeptide closely resembles theXenopus XFGF-CX protein, which was shown previously to be specificallyexpressed in highly proliferative tissues (see, e.g., Koga et al.,above). Therefore, it is presumed that FGF-CX would also modulatecellular activity in highly proliferative tissues. FGF-CX may thus beparticularly useful in diagnosing proliferative disorders and instimulating the growth of cells and tissues in order to overcomepathological states in which such growth has been suppressed orinhibited. Oligonucleotides corresponding to any one portion of theFGF-CX nucleic acids of SEQ ID NO: 1 may be used to detect theexpression of a FGF-CX-like gene. The proteins of the invention may beused to stimulate production of antibodies specifically binding theproteins. Such antibodies may be used in immunodiagnostic procedures todetect the occurrence of the protein in a sample. The proteins of theinvention may be used to stimulate cell growth and cell proliferation inconditions in which such growth would be favorable. An example would beto counteract toxic side effects of chemotherapeutic agents on, forexample, hematopoiesis and platelet formation, linings of thegastrointestinal tract, and hair follicles. They may also be used tostimulate new cell growth in neurological disorders including, forexample, Alzheimer's disease. Alternatively, antagonistic treatments maybe administered in which an antibody specifically binding the FGF-CX-like proteins of the invention would abrogate the specificgrowth-inducing effects of the proteins. Such antibodies may be useful,for example, in the treatment of proliferative disorders includingvarious tumors and benign hyperplasias.

[0297] An exemplary method for detecting the presence or absence ofFGF-CX in a biological sample involves obtaining a biological samplefrom a test subject and contacting the biological sample with a compoundor an agent capable of detecting FGF-CX protein or nucleic acid (e.g.,mRNA, genomic DNA) that encodes FGF-CX protein such that the presence ofFGF-CX is detected in the biological sample. An agent for detectingFGF-CX mRNA or genomic DNA is a labeled nucleic acid probe capable ofhybridizing to FGF-CX mRNA or genomic DNA. The nucleic acid probe canbe, for example, a full-length FGF-CX nucleic acid, such as the nucleicacid of SEQ ID NO: 1, or a portion thereof, such as an oligonucleotideof at least 15, 30, 50, 100, 250 or 500 nucleotides in length andsufficient to specifically hybridize under stringent conditions toFGF-CX mRNA or genomic DNA, as described above. Other suitable probesfor use in the diagnostic assays of the invention are described herein.

[0298] An agent for detecting FGF-CX protein is an antibody capable ofbinding to FGF-CX protein, preferably an antibody with a detectablelabel. Antibodies can be polyclonal, or more preferably, monoclonal. Anintact antibody, or a fragment thereof (e.g., Fab or F(ab′)₂) can beused. The term “labeled”, with regard to the probe or antibody, isintended to encompass direct labeling of the probe or antibody bycoupling (i.e., physically linking) a detectable substance to the probeor antibody, as well as indirect labeling of the probe or antibody byreactivity with another reagent that is directly labeled. Examples ofindirect labeling include detection of a primary antibody using afluorescently labeled secondary antibody and end-labeling of a DNA probewith biotin such that it can be detected with fluorescently labeledstreptavidin. The term “biological sample” is intended to includetissues, cells and biological fluids isolated from a subject, as well astissues, cells and fluids present within a subject. That is, thedetection method of the invention can be used to detect FGF-CX mRNA,protein, or genomic DNA in a biological sample in vitro as well as invivo. For example, in vitro techniques for detection of FGF-CX mRNAinclude Northern hybridizations and in situ hybridizations. In vitrotechniques for detection of FGF-CX protein include enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitations andimmunofluorescence. In vitro techniques for detection of FGF-CX genomicDNA include Southern hybridizations. Furthermore, in vivo techniques fordetection of FGF-CX protein include introducing into a subject a labeledanti-FGF-CX antibody. For example, the antibody can be labeled with aradioactive marker whose presence and location in a subject can bedetected by standard imaging techniques.

[0299] In one embodiment, the biological sample contains proteinmolecules from the test subject. Alternatively, the biological samplecan contain mRNA molecules from the test subject or genomic DNAmolecules from the test subject. A preferred biological sample is aperipheral blood leukocyte sample isolated by conventional means from asubject.

[0300] In another embodiment, the methods further involve obtaining acontrol biological sample from a control subject, contacting the controlsample with a compound or agent capable of detecting FGF-CX protein,mRNA, or genomic DNA, such that the presence of FGF-CX protein, mRNA orgenomic DNA is detected in the biological sample, and comparing thepresence of FGF-CX protein, mRNA or genomic DNA in the control samplewith the presence of FGF-CX protein, mRNA or genomic DNA in the testsample.

[0301] The invention also encompasses kits for detecting the presence ofFGF-CX in a biological sample. For example, the kit can comprise: alabeled compound or agent capable of detecting FGF-CX protein or mRNA ina biological sample; means for determining the amount of FGF-CX in thesample; and means for comparing the amount of FGF-CX in the sample witha standard. The compound or agent can be packaged in a suitablecontainer. The kit can further comprise instructions for using the kitto detect FGF-CX protein or nucleic acid.

[0302] Prognostic Assays

[0303] The diagnostic methods described herein can furthermore beutilized to identify subjects having or at risk of developing a diseaseor disorder associated with aberrant FGF-CX expression or activity. Forexample, the assays described herein, such as the preceding diagnosticassays or the following assays, can be utilized to identify a subjecthaving or at risk of developing a disorder associated with FGF-CXprotein, nucleic acid expression or activity in, e.g., proliferative ordifferentiative disorders such as hyperplasias, tumors, restenosis,psoriasis, Dupuytren's contracture, diabetic complications, orrheumatoid arthritis, etc.; and glia-associated disorders such ascerebral lesions, diabetic neuropathies, cerebral edema, seniledementia, Alzheimer's disease, etc. Alternatively, the prognostic assayscan be utilized to identify a subject having or at risk for developing adisease or disorder. Thus, the present invention provides a method foridentifying a disease or disorder associated with aberrant FGF-CXexpression or activity in which a test sample is obtained from a subjectand FGF-CX protein or nucleic acid (e.g., mRNA, genomic DNA) isdetected, wherein the presence of FGF-CX protein or nucleic acid isdiagnostic for a subject having or at risk of developing a disease ordisorder associated with aberrant FGF-CX expression or activity. As usedherein, a “test sample” refers to a biological sample obtained from asubject of interest. For example, a test sample can be a biologicalfluid (e.g., serum), cell sample, or tissue.

[0304] Furthermore, the prognostic assays described herein can be usedto determine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with aberrant FGF-CX expression or activity. For example,such methods can be used to determine whether a subject can beeffectively treated with an agent for a disorder, such as aproliferative disorder, differentiative disorder, glia-associateddisorders, etc. Thus, the present invention provides methods fordetermining whether a subject can be effectively treated with an agentfor a disorder associated with aberrant FGF-CX expression or activity inwhich a test sample is obtained and FGF-CX protein or nucleic acid isdetected (e.g., wherein the presence of FGF-CX protein or nucleic acidis diagnostic for a subject that can be administered the agent to treata disorder associated with aberrant FGF-CX expression or activity.)

[0305] The methods of the invention can also be used to detect geneticlesions in a FGF-CX gene, thereby determining if a subject with thelesioned gene is at risk for, or suffers from, a proliferative disorder,differentiative disorder, glia-associated disorder, etc. In variousembodiments, the methods include detecting, in a sample of cells fromthe subject, the presence or absence of a genetic lesion characterizedby at least one of an alteration affecting the integrity of a geneencoding a FGF-CX-protein, or the mis-expression of the FGF-CX gene. Forexample, such genetic lesions can be detected by ascertaining theexistence of at least one of (1) a deletion of one or more nucleotidesfrom a FGF-CX gene; (2) an addition of one or more nucleotides to aFGF-CX gene; (3) a substitution of one or more nucleotides of a FGF-CXgene, (4) a chromosomal rearrangement of a FGF-CX gene; (5) analteration in the level of a messenger RNA transcript of a FGF-CX gene,(6) aberrant modification of a FGF-CX gene, such as of the methylationpattern of the genomic DNA, (7) the presence of a non-wild type splicingpattern of a messenger RNA transcript of a FGF-CX gene, (8) a non-wildtype level of a FGF-CX-protein, (9) allelic loss of a FGF-CX gene, and(10) inappropriate post-translational modification of a FGF-CX-protein.As described herein, there are a large number of assay techniques knownin the art which can be used for detecting lesions in a FGF-CX gene. Apreferred biological sample is a peripheral blood leukocyte sampleisolated by conventional means from a subject. However, any biologicalsample containing nucleated cells may be used, including, for example,buccal mucosal cells.

[0306] In certain embodiments, detection of the lesion involves the useof a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S.Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) PNAS91:360-364), the latter of which can be particularly useful fordetecting point mutations in the FGF-CX-gene (see Abravaya et al. (1995)Nucl Acids Res 23:675-682). This method can include the steps ofcollecting a sample of cells from a patient, isolating nucleic acid(e.g., genomic, mRNA or both) from the cells of the sample, contactingthe nucleic acid sample with one or more primers that specificallyhybridize to a FGF-CX gene under conditions such that hybridization andamplification of the FGF-CX gene (if present) occurs, and detecting thepresence or absence of an amplification product, or detecting the sizeof the amplification product and comparing the length to a controlsample. It is anticipated that PCR and/or LCR may be desirable to use asa preliminary amplification step in conjunction with any of thetechniques used for detecting mutations described herein.

[0307] Alternative amplification methods include: self sustainedsequence replication (Guatelli et al., 1990, Proc Natl Acad Sci USA87:1874-1878), transcriptional amplification system (Kwoh, et al., 1989,Proc Natl Acad Sci USA 86:1173-1177), Q-Beta Replicase (Lizardi et al,1988, Bio Technology 6:1197), or any other nucleic acid amplificationmethod, followed by the detection of the amplified molecules usingtechniques well known to those of skill in the art. These detectionschemes are especially useful for the detection of nucleic acidmolecules if such molecules are present in very low numbers.

[0308] In an alternative embodiment, mutations in a FGF-CX gene from asample cell can be identified by alterations in restriction enzymecleavage patterns. For example, sample and control DNA is isolated,amplified (optionally), digested with one or more restrictionendonucleases, and fragment length sizes are determined by gelelectrophoresis and compared. Differences in fragment length sizesbetween sample and control DNA indicates mutations in the sample DNA.Moreover, the use of sequence specific ribozymes (see, for example, U.S.Pat. No. 5,493,531) can be used to score for the presence of specificmutations by development or loss of a ribozyme cleavage site.

[0309] In other embodiments, genetic mutations in FGF-CX can beidentified by hybridizing a sample and control nucleic acids, e.g., DNAor RNA, to high density arrays containing hundreds or thousands ofoligonucleotides probes (Cronin et al. (1996) Human Mutation 7: 244-255;Kozal et al. (1996) Nature Medicine 2: 753-759). For example, geneticmutations in FGF-CX can be identified in two dimensional arrayscontaining light-generated DNA probes as described in Cronin et al.above. Briefly, a first hybridization array of probes can be used toscan through long stretches of DNA in a sample and control to identifybase changes between the sequences by making linear arrays of sequentialoverlapping probes. This step allows the identification of pointmutations. This step is followed by a second hybridization array thatallows the characterization of specific mutations by using smaller,specialized probe arrays complementary to all variants or mutationsdetected. Each mutation array is composed of parallel probe sets, onecomplementary to the wild-type gene and the other complementary to themutant gene.

[0310] In yet another embodiment, any of a variety of sequencingreactions known in the art can be used to directly sequence the FGF-CXgene and detect mutations by comparing the sequence of the sample FGF-CXwith the corresponding wild-type (control) sequence. Examples ofsequencing reactions include those based on techniques developed byMaxim and Gilbert (1977) PNAS 74:560 or Sanger (1977) PNAS 74:5463. Itis also contemplated that any of a variety of automated sequencingprocedures can be utilized when performing the diagnostic assays (Naeveet al., (1995) Biotechniques 19:448), including sequencing by massspectrometry (see, e.g., PCT International Publ. No. WO 94/16101; Cohenet al. (1996)Adv Chromatogr 36:127-162; and Griffin et al. (1993) ApplBiochem Biotechnol 38:147-159).

[0311] Other methods for detecting mutations in the FGF-CX gene includemethods in which protection from cleavage agents is used to detectmismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al.(1985) Science 230:1242). In general, the art technique of “mismatchcleavage” starts by providing heteroduplexes of formed by hybridizing(labeled) RNA or DNA containing the wild-type FGF-CX sequence withpotentially mutant RNA or DNA obtained from a tissue sample. Thedouble-stranded duplexes are treated with an agent that cleavessingle-stranded regions of the duplex such as which will exist due tobasepair mismatches between the control and sample strands. Forinstance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybridstreated with S1 nuclease to enzymatically digesting the mismatchedregions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can betreated with hydroxylamine or osmium tetroxide and with piperidine inorder to digest mismatched regions. After digestion of the mismatchedregions, the resulting material is then separated by size on denaturingpolyacrylamide gels to determine the site of mutation. See, for example,Cotton et al (1988) Proc Natl Acad Sci USA 85:4397; Saleeba et al (1992)Methods Enzymol 217:286-295. In an embodiment, the control DNA or RNAcan be labeled for detection.

[0312] In still another embodiment, the mismatch cleavage reactionemploys one or more proteins that recognize mismatched base pairs indouble-stranded DNA (so called “DNA mismatch repair” enzymes) in definedsystems for detecting and mapping point mutations in FGF-CX cDNAsobtained from samples of cells. For example, the mutY enzyme of E. colicleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLacells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis15:1657-1662). According to an exemplary embodiment, a probe based on aFGF-CX sequence, e.g., a wild-type FGF-CX sequence, is hybridized to acDNA or other DNA product from a test cell(s). The duplex is treatedwith a DNA mismatch repair enzyme, and the cleavage products, if any,can be detected from electrophoresis protocols or the like. See, forexample, U.S. Pat. No. 5,459,039.

[0313] In other embodiments, alterations in electrophoretic mobilitywill be used to identify mutations in FGF-CX genes. For example, singlestrand conformation polymorphism (SSCP) may be used to detectdifferences in electrophoretic mobility between mutant and wild typenucleic acids (Orita et al (1989) Proc Natl Acad Sci USA: 86:2766, seealso Cotton (1993) Mutat Res 285:125-144; Hayashi (1992) Genet Anal TechAppl 9:73-79). Single-stranded DNA fragments of sample and controlFGF-CX nucleic acids will be denatured and allowed to renature. Thesecondary structure of single-stranded nucleic acids varies according tosequence, the resulting alteration in electrophoretic mobility enablesthe detection of even a single base change. The DNA fragments may belabeled or detected with labeled probes. The sensitivity of the assaymay be enhanced by using RNA, rather than DNA, in which the secondarystructure is more sensitive to a change in sequence. In one embodiment,the subject method utilizes heteroduplex analysis to separate doublestranded heteroduplex molecules on the basis of changes inelectrophoretic mobility. See, e.g., Keen et al. (1991) Trends Genet7:5.

[0314] In yet another embodiment the movement of mutant or wild-typefragments in polyacrylamide gels containing a gradient of denaturant isassayed using denaturing gradient gel electrophoresis (DGGE). See, e.g.,Myers et al (1985) Nature 313:495. When DGGE is used as the method ofanalysis, DNA will be modified to insure that it does not completelydenature, for example by adding a GC clamp of approximately 40 bp ofhigh-melting GC-rich DNA by PCR. In a further embodiment, a temperaturegradient is used in place of a denaturing gradient to identifydifferences in the mobility of control and sample DNA. See, e.g.,Rosenbaum and Reissner (1987) Biophys Chem 265:12753.

[0315] Examples of other techniques for detecting point mutationsinclude, but are not limited to, selective oligonucleotidehybridization, selective amplification, or selective primer extension.For example, oligonucleotide primers may be prepared in which the knownmutation is placed centrally and then hybridized to target DNA underconditions that permit hybridization only if a perfect match is found.See, e.g., Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) ProcNatl Acad. Sci USA 86:6230. Such allele specific oligonucleotides arehybridized to PCR amplified target DNA or a number of differentmutations when the oligonucleotides are attached to the hybridizingmembrane and hybridized with labeled target DNA.

[0316] Alternatively, allele specific amplification technology thatdepends on selective PCR amplification may be used in conjunction withthe instant invention. Oligonucleotides used as primers for specificamplification may carry the mutation of interest in the center of themolecule (so that amplification depends on differential hybridization)(Gibbs et al. (1989) Nucleic Acids Res 17:2437-2448) or at the extreme3′ end of one primer where, under appropriate conditions, mismatch canprevent, or reduce polymerase extension (Prossner (1993) Tibtech11:238). In addition it may be desirable to introduce a novelrestriction site in the region of the mutation to create cleavage-baseddetection. See, e.g., Gasparini et al (1992) Mol Cell Probes 6:1. It isanticipated that in certain embodiments amplification may also beperformed using Taq ligase for amplification. See, e.g., Barany (1991)Proc Natl Acad Sci USA 88:189. In such cases, ligation will occur onlyif there is a perfect match at the 3′ end of the 5′ sequence, making itpossible to detect the presence of a known mutation at a specific siteby looking for the presence or absence of amplification.

[0317] The methods described herein may be performed, for example, byutilizing pre-packaged diagnostic kits comprising at least one probenucleic acid or antibody reagent described herein, which may beconveniently used, e.g., in clinical settings to diagnose patientsexhibiting symptoms or family history of a disease or illness involvinga FGF-CX gene.

[0318] Furthermore, any cell type or tissue, preferably peripheral bloodleukocytes, in which FGF-CX is expressed may be utilized in theprognostic assays described herein. However, any biological samplecontaining nucleated cells may be used, including, for example, buccalmucosal cells.

[0319] Pharmacogenomics

[0320] Agents, or modulators that have a stimulatory or inhibitoryeffect on FGF-CX activity (e.g., FGF-CX gene expression), as identifiedby a screening assay described herein can be administered to individualsto treat (prophylactically or therapeutically) disorders (e.g.,neurological, cancer-related or gestational disorders) associated withaberrant FGF-CX activity. In conjunction with such treatment, thepharmacogenomics (i.e., the study of the relationship between anindividual's genotype and that individual's response to a foreigncompound or drug) of the individual may be considered. Differences inmetabolism of therapeutics can lead to severe toxicity or therapeuticfailure by altering the relation between dose and blood concentration ofthe pharmacologically active drug. Thus, the pharmacogenomics of theindividual permits the selection of effective agents (e.g., drugs) forprophylactic or therapeutic treatments based on a consideration of theindividual's genotype. Such pharmacogenomics can further be used todetermine appropriate dosages and therapeutic regimens. Accordingly, theactivity of FGF-CX protein, expression of FGF-CX nucleic acid, ormutation content of FGF-CX genes in an individual can be determined tothereby select appropriate agent(s) for therapeutic or prophylactictreatment of the individual.

[0321] Pharmacogenomics deals with clinically significant hereditaryvariations in the response to drugs due to altered drug disposition andabnormal action in affected persons. See e.g., Eichelbaum, 1996, ClinExp Pharmacol Physiol, 23:983-985 and Linder, 1997, Clin Chem,43:254-266. In general, two types of pharmacogenetic conditions can bedifferentiated. Genetic conditions transmitted as a single factoraltering the way drugs act on the body (altered drug action) or geneticconditions transmitted as single factors altering the way the body actson drugs (altered drug metabolism). These pharmacogenetic conditions canoccur either as rare defects or as polymorphisms. For example,glucose-6-phosphate dehydrogenase (G6PD) deficiency is a commoninherited enzymopathy in which the main clinical complication ishaemolysis after ingestion of oxidant drugs (anti-malarials,sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

[0322] As an illustrative embodiment, the activity of drug metabolizingenzymes is a major determinant of both the intensity and duration ofdrug action. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, PM show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

[0323] Thus, the activity of FGF-CX protein, expression of FGF-CXnucleic acid, or mutation content of FGF-CX genes in an individual canbe determined to thereby select appropriate agent(s) for therapeutic orprophylactic treatment of the individual. In addition, pharmacogeneticstudies can be used to apply genotyping of polymorphic alleles encodingdrug-metabolizing enzymes to the identification of an individual's drugresponsiveness phenotype. This knowledge, when applied to dosing or drugselection, can avoid adverse reactions or therapeutic failure and thusenhance therapeutic or prophylactic efficiency when treating a subjectwith a FGF-CX modulator, such as a modulator identified by one of theexemplary screening assays described herein.

[0324] Monitoring Clinical Efficacy

[0325] Monitoring the influence of agents (e.g., drugs, compounds) onthe expression or activity of FGF-CX (e.g., the ability to modulateaberrant cell proliferation and/or differentiation) can be applied inbasic drug screening and in clinical trials. For example, theeffectiveness of an agent determined by a screening assay as describedherein to increase FGF-CX gene expression, protein levels, or upregulateFGF-CX activity, can be monitored in clinical trials of subjectsexhibiting decreased FGF-CX gene expression, protein levels, ordownregulated FGF-CX activity. Alternatively, the effectiveness of anagent determined by a screening assay to decrease FGF-CX geneexpression, protein levels, or downregulate FGF-CX activity, can bemonitored in clinical trials of subjects exhibiting increased FGF-CXgene expression, protein levels, or upregulated FGF-CX activity. In suchclinical trials, the expression or activity of FGF-CX and, preferably,other genes that have been implicated in, for example, a proliferativeor neurological disorder, can be used as a “read out” or marker of theresponsiveness of a particular cell.

[0326] For example, genes, including FGF-CX, that are modulated in cellsby treatment with an agent (e.g., compound, drug or small molecule) thatmodulates FGF-CX activity (e.g., identified in a screening assay asdescribed herein) can be identified. Thus, to study the effect of agentson cellular proliferation disorders, for example, in a clinical trial,cells can be isolated and RNA prepared and analyzed for the levels ofexpression of FGF-CX and other genes implicated in the disorder. Thelevels of gene expression (i.e., a gene expression pattern) can bequantified by Northern blot analysis or RT-PCR, as described herein, oralternatively by measuring the amount of protein produced, by one of themethods as described herein, or by measuring the levels of activity ofFGF-CX or other genes. In this way, the gene expression pattern canserve as a marker, indicative of the physiological response of the cellsto the agent. Accordingly, this response state may be determined before,and at various points during, treatment of the individual with theagent.

[0327] In one embodiment, the invention provides a method for monitoringthe effectiveness of treatment of a subject with an agent (e.g., anagonist, antagonist, protein, peptide, nucleic acid, peptidomimetic,small molecule, or other drug candidate identified by the screeningassays described herein) comprising the steps of (i) obtaining apre-administration sample from a subject prior to administration of theagent; (ii) detecting the level of expression of a FGF-CX protein, mRNA,or genomic DNA in the preadministration sample; (iii) obtaining one or30 more post-administration samples from the subject; (iv) detecting thelevel of expression or activity of the FGF-CX protein, mRNA, or genomicDNA in the post-administration samples; (v) comparing the level ofexpression or activity of the FGF-CX protein, mRNA, or genomic DNA inthe pre-administration sample with the FGF-CX protein, mRNA, or genomicDNA in the post administration sample or samples; and (vi) altering theadministration of the agent to the subject accordingly. For example,increased administration of the agent may be desirable to increase theexpression or activity of FGF-CX to higher levels than detected, i.e.,to increase the effectiveness of the agent. Alternatively, decreasedadministration of the agent may be desirable to decrease expression oractivity of FGF-CX to lower levels than detected, i.e., to decrease theeffectiveness of the agent.

[0328] Methods of Treatment

[0329] The present invention provides for both prophylactic andtherapeutic methods of treating a subject at risk of (or susceptible to)a disorder or having a disorder associated with aberrant FGF-CXexpression or activity.

[0330] Diseases and disorders that are characterized by increased(relative to a subject not suffering from the disease or disorder)levels or biological activity may be treated with Therapeutics thatantagonize (i.e., reduce or inhibit) activity. Therapeutics thatantagonize activity may be administered in a therapeutic or prophylacticmanner. Therapeutics that may be utilized include, but are not limitedto, (i) a FGF-CX polypeptide, or analogs, derivatives, fragments orhomologs thereof; (ii) antibodies to a FGF-CX peptide; (iii) nucleicacids encoding a FGF-CX peptide; (iv) administration of antisensenucleic acid and nucleic acids that are “dysfunctional” (i.e., due to aheterologous insertion within the coding sequences of coding sequencesto a FGF-CX peptide) that are utilized to “knockout” endogenous functionof a FGF-CX peptide by homologous recombination (see, e.g., Capecchi,1989, Science 244: 1288-1292); or (v) modulators (i.e., inhibitors,agonists and antagonists, including additional peptide mimetic of theinvention or antibodies specific to a peptide of the invention) thatalter the interaction between a FGF-CX peptide and its binding partner.

[0331] Diseases and disorders that are characterized by decreased(relative to a subject not suffering from the disease or disorder)levels or biological activity may be treated with Therapeutics thatincrease (i.e., are agonists to) activity. Therapeutics that upregulateactivity may be administered in a therapeutic or prophylactic manner.Therapeutics that may be utilized include, but are not limited to, aFGF-CX peptide, or analogs, derivatives, fragments or homologs thereof;or an agonist that increases bioavailability.

[0332] Increased or decreased levels can be readily detected byquantifying peptide and/or RNA, by obtaining a patient tissue sample(e.g., from biopsy tissue) and assaying it in vitro for RNA or peptidelevels, structure and/or activity of the expressed peptides (or mRNAs ofa FGF-CX peptide). Methods that are well-known within the art include,but are not limited to, immunoassays (e.g., by Western blot analysis,immunoprecipitation followed by sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/orhybridization assays to detect expression of mRNAs (e.g., Northernassays, dot blots, in situ hybridization, etc.).

[0333] In one aspect, the invention provides a method for preventing, ina subject, a disease or condition associated with an aberrant FGF-CXexpression or activity, by administering to the subject an agent thatmodulates FGF-CX expression or at least one FGF-CX activity. Subjects atrisk for a disease that is caused or contributed to by aberrant FGF-CXexpression or activity can be identified by, for example, any or acombination of diagnostic or prognostic assays as described herein.Administration of a prophylactic agent can occur prior to themanifestation of symptoms characteristic of the FGF-CX aberrancy, suchthat a disease or disorder is prevented or, alternatively, delayed inits progression. Depending on the type of FGF-CX aberrancy, for example,a FGF-CX agonist or FGF-CX antagonist agent can be used for treating thesubject. The appropriate agent can be determined based on screeningassays described herein.

[0334] Another aspect of the invention pertains to methods of modulatingFGF-CX expression or activity for therapeutic purposes. The modulatorymethod of the invention involves contacting a cell with an agent thatmodulates one or more of the activities of FGF-CX protein activityassociated with the cell. An agent that modulates FGF-CX proteinactivity can be an agent as described herein, such as a nucleic acid ora protein, a naturally-occurring cognate ligand of a FGF-CX protein, apeptide, a FGF-CX peptidomimetic, or other small molecule. In oneembodiment, the agent stimulates one or more FGF-CX protein activity.Examples of such stimulatory agents include active FGF-CX protein and anucleic acid molecule encoding FGF-CX that has been introduced into thecell. In another embodiment, the agent inhibits one or more FGF-CXprotein activity. Examples of such inhibitory agents include antisenseFGF-CX nucleic acid molecules and anti-FGF-CX antibodies. Thesemodulatory methods can be performed in vitro (e.g., by culturing thecell with the agent) or, alternatively, in vivo (e.g., by administeringthe agent to a subject). As such, the present invention provides methodsof treating an individual afflicted with a disease or disordercharacterized by aberrant expression or activity of a FGF-CX protein ornucleic acid molecule. In one embodiment, the method involvesadministering an agent (e.g., an agent identified by a screening assaydescribed herein), or combination of agents that modulates (e.g.,upregulates or downregulates) FGF-CX expression or activity. In anotherembodiment, the method involves administering a FGF-CX protein ornucleic acid molecule as therapy to compensate for reduced or aberrantFGF-CX expression or activity.

[0335] The invention will be further illustrated in the followingnon-limiting examples.

EXAMPLES Example 1 Identification of the FGF-CX Gene

[0336] The FGF-CX gene was identified following a TBLASTN (Altschul, etal (1990) J. Mol. Biol. 215, 403-410) search of Genbank human genomicDNA sequences with Xenopus FGF-CX (Koga, et al. (1999) Biochem. Biophys.Res. Comm. 261, 756-765; Accession No. AB012615) as query. This searchidentified a locus (Accession No. AB020858) of high homology onchromosome 8. Intron/exon boundaries were deduced using standardconsensus splicing parameters (Mount (1996) Science 271, 1690-1692),together with homologies derived from known FGFs. The FGF-CX initiationcodon localizes to bp 16214 of the sequence of AB020858, and theremaining 3′ portion of this exon continues to bp 15930. The 5′ UTR ofFGF-CX was extended upstream of the initiation codon by an additional606 bp using public ESTs (Accession Nos. AA232729, AA236522, AI272876and AI272878). The remaining structure of the FGF-CX gene as it relatesto locus AB020858 is as follows: intron 1 (bp 15929-9942); exon 2 (bp9941-9838); intron 2 (bp 9837-7500); exon 3 (begins at bp 7499 andcontinues as shown in Table 13; the structure of the 3′ untranslatedregion has not yet been determined). Table 13 presents an analysis ofthe FGF-CX gene, including the nucleotide (SEQ ID NO: 25) and deducedamino acid (SEQ ID NO: 2) sequence of FGF-CX. The initiation and stopcodons are in bold, and an in frame stop codon residing in the 5′ UTR isunderlined. TABLE 13    Exon 1...AGACAGTGAGAGCTTCCCTGCCATTTCAGTGCAAAGTCCCTCCGGAGCGACCTCAGAGGAGTAACCGGGCCTTAACTTTTTGCGCTCGTTTTGCTATAATTTTTCTCTATCCACCTCCATCCCACCCCCACAACACTCTTTACTGGGGGGGTCTTTTGTGTTCCGGATCTCCCCCTCCATGGCTCCCTTAGCCGAAGTCGGGGGCTTTCTGGGCGGCCTGGAGGGCTTGGGCCAGCA1                     M  A  P  L  A  E  V  G  G  F  L  G  G  L  E  G  L  G  Q  QGGTGGGTTCGCATTTCCTGTTGCCTCCTGCCGGGGAGCGGCCGCCGCTGCTGGGCGAGCGCAGGAGCGCGGCGGAGCGGA21 V  G  S  H  F  L  L  P  P  A  G  E  R  P  P  L  L  G  E  R  R  S  A  A  E  R  SGCGCGCGCGGCGGGCCGGGGGCTGCGCAGCTGGCGCACCTGCACGGCATCCTGCGCCGCCGGCAGCTCTATTGCCGCACC48  A  R  G  G  P  G  A  A  Q  L  A  H  L  H  G  I  L  R  R  R  Q  L  Y  C  R  T                                                           <-|-> Exon 2GGCTTCCACCTGCAGATCCTGCCCGACGGCAGCGTGCAGGGCACCCGGCAGGACCACAGCCTCTTCGGTATCTTGGAATT74G  F  H  L  Q  I  L  P  D  G  S  V  Q  G  T  R  Q  D  H  S  L  F  G  I  L  H  FCATCAGTGTGGCAGTGGGACTGGTCAGTATTAGAGGTGTGGACAGTGGTCTCTATCTTGGAATGAATGACAAAGGAGAAC101 I  S  V  A  V  G  L  V  S  I  R  G  V  D  S  G  L  Y  L  G  M  N  D  K  G  E  L       <-|-> Exon 3TCTATGGATCAGAGAAACTTACTTCCGAATGCATCTTTAGGGAGCAGTTTGAAGAGAACTGGTATAACACCTATTCATCT128  Y  G  S  E  K  L  T  S  E  C  I  F  R  E  Q  F  E  E  N  W  Y  N  T  Y  S  SAACATATATAAACATGGAGACACTGGCCGCAGGTATTTTGTGGCACTTAACAAAGACGGAACTCCAAGAGATGGCGCCAG154N  I  Y  K  H  G  D  T  G  R  R  Y  F  V  A  L  N  K  D  G  T  P  R  D  G  A  RGTCCAAGAGGCATCAGAAATTTACACATTTCTTACCTAGACCAGTGGATCCAGAAAGAGTTCCAGAATTGTACAAGGACC181 S  K  R  H  Q  K  F  T  H  F  L  P  R  P  V  D  P  E  R  V  P  E  L  Y  K  D  LTACTGATGTACACTTGA... 208   L  M  Y  T

[0337] The gene discovered by the procedure in the preceding paragraphincludes 3 exons and 2 introns (Table 13). The DNA sequence predicts anORF of 211 amino acid residues, with an in-frame stop codon 117 bpupstream of the initiator methionine. The DNA segment from which thegene was mined maps to chromosome 8p21.3-p22, a location that wasconfirmed by radiation hybrid analysis (see Example 2).

[0338] An FGF signature motif,G-X-[LI]-X-[STAGP]-X(6,7)-[DE]-C-X-[FLM]-X-E-X(6)-Y, identified by aPROSITE search (Bucher & Bairoch (1994) Ismb. 2, 53-61) located betweenamino acid residues 125-148 is double-underlined, and intron/exonboundaries are depicted with arrows. Introns 1 and 2 are 5988 bp and2338 bp long, respectively. The 5′ UTR sequence was derived from publicESTs, and is not shown in its entirety.

Example 2 Radiation Hybrid Mapping of FGF-CX

[0339] Radiation hybrid mapping using human chromosome markers wascarried out for FGF-CX. The procedure used is analogous to thatdescribed in Steen, RG et al. (A High-Density Integrated Genetic Linkageand Radiation Hybrid Map of the Laboratory Rat, Genome Research 1999(Published Online on May 21, 1999)Vol. 9, AP1-AP8, 1999). A panel of 93cell clones containing the randomized radiation-induced humanchromosomal fragments was screened in 96 well plates using PCR primersdesigned to identify the sought clones in a unique fashion. The DNAsegment from which the nucleotide sequence encoding FGF-CX wasidentified was annotated as mapping to chromosome 8p21.3-p22. Thisresult was refined by the present analysis by finding that FGF-CX mapsto chromosome 8 at a locus which overlaps marker AFM177XB10, and whichis 1.6 cR from marker WI-5104 and 3.2 cR from marker WI-9262.

Example 3 Molecular Cloning of the Sequence Encoding a FGF-CX Protein

[0340] Oligonucleotide primers were designed for the amplification byPCR of a DNA segment, representing an open reading frame, coding for thefull length FGF-CX. The forward primer includes a BglII restriction site(AGATCT) and a consensus Kozak sequence (CCACC). The reverse primercontains an in-frame XhoI restriction site for further subcloningpurposes. Both the forward and the reverse primers contain a 5′ clampsequence (CTCGTC). The sequences of the primers are the following:FGF-CX-Forward: 5′ - CTCGTC AGATCT CCACC ATG GCT CCC TTA GCC GAA GTC -3′ (SEQ ID NO: 3) FGF-CX-Reverse: 5′ - CTCGTC CTCGAG AGT GTA CAT CAG TAGGTC CTT G - 3′ (SEQ ID NO: 4)

[0341] PCR reactions were performed using a total of 5 ng human prostatecDNA template, 1 μM of each of the FGF-CX-Forward and FGF-CX-Reverseprimers, 5 micromoles dNTP (Clontech Laboratories, Palo Alto Calif.) and1 microliter of 50×Advantage-HF 2 polymerase (Clontech Laboratories) in50 microliter volume. The following PCR reaction conditions were used:

[0342] a) 96° C. 3 minutes

[0343] b) 96° C. 30 seconds denaturation

[0344] c) 70° C. 30 seconds, primer annealing. This temperature wasgradually decreased by 1° C./cycle.

[0345] d) 72° C. 1 minute extension.

[0346] Repeat steps (b)-(d) ten times

[0347] e) 96° C. 30 seconds denaturation

[0348] f) 60° C. 30 seconds annealing

[0349] g) 72° C. 1 minute extension

[0350] Repeat steps (e)-(g) 25 times

[0351] h) 72° C. 5 minutes final extension

[0352] A single PCR product, with the expected size of approximately 640bp, was isolated after electrophoresis on agarose gel and ligated into apCR2.1 vector (Invitrogen, Carlsbad, Calif.). The cloned insert wassequenced using vector specific M13 Forward(−40) and M13 Reverseprimers, which verified that the nucleotide sequence was 100% identicalto the sequence in Table 1 (SEQ ID NO: 1) inserted directly between theupstream BglII cloning site and the downstream XhoI cloning site. Thecloned sequence constitutes an open reading frame coding for thepredicted FGF-CX full length protein. The clone is calledTA-AB02085-S274-F19.

Example 4 Preparation of Mammalian Expression Vector pCEP4/Sec

[0353] The oligonucleotide primers pSec-V5-His Forward (CTCGT CCTCGAGGGT AAGCC TATCC CTAAC (SEQ ID NO: 14)) and pSec-V5-His Reverse (CTCGTCGGGC CCCTG ATCAG CGGGT TTAAA C (SEQ ID NO: 15)), were designed toamplify a fragment from the pcDNA3.1-V5His (Invitrogen, Carlsbad, CA)expression vector that includes V5 and His6. The PCR product wasdigested with XhoI and ApaI and ligated into the XhoI/ApaI digestedpSecTag2 B vector harboring an Ig kappa leader sequence (Invitrogen,Carlsbad Calif.). The correct structure of the resulting vector,pSecV5His, including an in-frame Ig-kappa leader and V5-His6 wasverified by DNA sequence analysis. The vector pSecV5His was digestedwith PmeI and NheI to provide a fragment retaining the above elements inthe correct frame. The PmeI-NheI fragment was ligated into theBamHI/Klenow and NheI treated vector pCEP4 (Invitrogen, Carlsbad,Calif.). The resulting vector was named pCEP4/Sec and includes anin-frame Ig kappa leader, a site for insertion of a clone of interest,and the V5 epitope and 6×His under control of the PCMV and/or the PT7promoter. pCEP4/Sec is an expression vector that allows heterologousprotein expression and secretion by fusing any protein into a multiplecloning site following the Ig kappa chain signal peptide. Detection andpurification of the expressed protein are aided by the presence of theV5 epitope tag and 6×His tag at the C-terminus (Invitrogen, Carlsbad,Calif.).

Example 5 Expression of FGF-CX in Human Embryonic Kidney (HEK) 293 Cells

[0354] The BglII-XhoI fragment containing the FGF-CX sequence wasisolated from TA-AB02085-S274-F19 (Example 3) and subcloned into theBamHI-XhoI digested pCEP4/Sec to generate the expression vectorpCEP4/Sec-FGF-CX. The pCEP4/Sec-FGF-CX vector was transfected into 293cells using the LipofectaminePlus reagent following the manufacturer'sinstructions (Gibco/BRL/Life Technologies, Rockville, Md.). The cellpellet and supernatant were harvested 72 hours after transfection andexamined for FGF-CX expression by Western blotting (reducing conditions)with an anti-V5 antibody. FIG. 2 shows that FGF-CX is expressed as apolypeptide having an apparent molecular weight (Mr) of approximately 34kDa proteins secreted by 293 cells. In addition a minor band is observedat about 31 kDa.

Example 6 Expression of FGF-CX in E. coli

[0355] The vector pRSETA (In Vitrogen Inc., Carlsbad, Calif.) wasdigested with XhoI and NcoI restriction enzymes. Oligonucleotide linkersof the sequence 5′ CATGGTCAGCCTAC 3′ (SEQ ID NO: 16) and 5′TCGAGTAGGCTGAC 3′ (SEQ ID NO: 17) were annealed at 37 degree Celsius andligated into the XhoI-NcoI treated pRSETA. The resulting vector wasconfirmed by restriction analysis and sequencing and was named pETMY.The BglII-XhoI fragment of the sequence encoding FGF-CX (see Example 3)was ligated into vector pETMY that was digested with BamHI and XhoIrestriction enzymes. The expression vector is named pETMY-FGF-CX. Inthis vector, hFGF-CX was fused to the 6×His tag and T7 epitope at itsN-terminus. The plasmid pETMY-FGF-CX was then transfected into the E.coli expression host BL21 (DE3, pLys) (Novagen, Madison, Wis.) andexpression of protein FGF-CX was induced according to the manufacturer'sinstructions. After induction, total cells were harvested, and proteinswere analyzed by Western blotting using anti-HisGly antibody(Invitrogen, Carlsbad, Calif.). FIG. 3 shows that FGF-CX was expressedas a protein of Mr approximately 32 kDa.

Example 7 Comparison of Expression of Recombinant FGF-CX Protein Withand Without a Cloned Signal Peptide

[0356] a) Expression Without a Signal Peptide

[0357] As noted in the Detailed Description of the Invention, FGF-CXapparently lacks a classical amino-terminal signal sequence. Todetermine whether FGF-CX is secreted from mammalian cells, cDNA obtainedas the BglII-XhoI fragment, encoding the full length FGF-CX protein, wassubcloned from TA-AB02085-S274-F19 (Example 3) into BamHI/XhoI-digestedpcDNA3.1 (Invitrogen). This provided a mammalian expression vectordesignated pFGF-CX. This construct incorporates the V5 epitope tag and apolyhistidine tag into the carboxy-terminus of the protein to aid in itsidentification and purification, respectively, and should generate apolypeptide of about 27 kDa. Following transient transfection into 293human embryonic kidney cells, conditioned media was harvested 48 hr posttransfection.

[0358] In addition to secretion of FGF-CX into conditioned media, italso found to be associated with the cell pellet/ECM (data not shown).Since FGFs are known to bind to heparin sulfate proteoglycan (HSPG)present on the surface of cells and in the extracellular matrix (ECM),the inventors investigated the possibility that FGF-CX was sequesteredin this manner. To this end, FGF-CX-transfected cells were extracted bytreatment with 0.5 ml DMEM containing 100 □M suramin, a compound knownto disrupt low affinity interactions between growth factors and HSPGs(La Rocca, R. V., Stein, C. A. & Myers, C. E. (1990) Cancer Cells 2,106-115), for 30 min at 4° C. The suramin-extracted conditioned mediawas then harvested and clarified by centrifigation (5 min; 2000×g).

[0359] The conditioned media and the suramin extract were then mixedwith equal volumes of 2×gel-loading buffer. Samples were boiled for 10min, resolved by SDS-PAGE on 4-20% gradient polyacrylamide gels (Novex,Dan Diego, Calif.) under reducing conditions, and transferred tonitrocelluose filters (Novex). Western analysis was performed accordingto standard procedures using HRP-conjugated anti-V5 antibody(Invitrogen) and the ECL detection system (Amersham Pharmacia Biotech,Piscataway, N.J.).

[0360] One band having the expected Mr was identified in conditionedmedia from 293 cells transfected with pFGF-CX (FIG. 1A, lane 1).Conditioned media from cells transfected with control vector did notreact with the antibody (FIG. 1A, lane 5). After suramin treatment, itwas found that a significant quantity of FGF-CX could in fact bereleased from the cell surface/ECM, indicating that HSPGs are likely toplay a role in sequestering this protein (FIG. 1A, lane 2). Theseresults indicate that FGF-CX can be secreted without a classical signalpeptide.

[0361] Recombinant FGF-CX protein stimulates DNA synthesis and cellproliferation, effects that are likely to be mediated via high affinitybinding of FGF-CX to a cell surface receptor, and modulated via lowaffinity interactions with HSPGs. The suramin extraction data suggeststhat FGF-CX binds to HSPGs present on the cell surface and/or the ECM.

[0362] b) Expression With a Signal Peptide

[0363] With the goal of enhancing protein secretion, a construct(pCEP4/Sec-FGF-CX) was generated in which the FGF-CX cDNA was fused inframe with a cleavable amino-terminal secretory signal sequence derivedfrom the Igκ gene. The resulting protein also contained carboxy-terminalV5 and polyhistidine tags as described above for pFGF-CX. Followingtransfection into 293 cells, a protein product having the expected Mr ofabout 31 kDa was obtained, and suramin was again found to release asignificant quantity of sequestered FGF-CX protein (FIG. 1A; lanes 3 and4). As expected, pCEP4/Sec-FGF-CX generated more soluble FGF-CX proteinthan did pFGF-CX.

[0364] Results similar to those described above for 293 cells were alsoobtained with NIH 3T3 cells (FIG. 1B).

Example 8 Real Time Quantitative Expression Analysis Of FGF-CX NucleicAcids By PCR

[0365] The quantitative expression of various clones was assessed in 41normal and 55 tumor samples (in most cases, the samples presented inFIG. 4, Panels A and B are those identified in Table 14) by real timequantitative PCR (TAQMAN® analysis) performed on a Perkin-ElmerBiosystems ABI PRISM® 7700 Sequence Detection System. In Table 14, thefollowing abbreviations are used:

[0366] ca.=carcinoma,

[0367] *=established from metastasis,

[0368] met=metastasis,

[0369] s cell var=small cell variant,

[0370] non-s =non-sm=non-small,

[0371] squam=squamous,

[0372] p1. eff=p1 effusion=pleural effusion,

[0373] glio=glioma,

[0374] astro=astrocytoma, and

[0375] neuro=neuroblastoma.

[0376] First, 96 RNA samples were normalized to β-actin andglyceraldehyde-3-phosphate dehydrogenase (GAPDH). RNA (˜50 ng total or˜1 ng polyA⁺) was converted to cDNA using the TAQMAN® ReverseTranscription Reagents Kit (PE Biosystems, Foster City, Calif.; cat#N808-0234) and random hexamers according to the manufacturer'sprotocol. Reactions were performed in 20 μl and incubated for 30 min. at48° C. cDNA (5 μl) was then transferred to a separate plate for theTAQMAN® reaction using β-actin and GAPDH TAQMAN® Assay Reagents (PEBiosystems; cat. no.'s 4310881E and 4310884E, respectively) and TAQMAN®universal PCR Master Mix (PE Biosystems; cat #4304447) according to themanufacturer's protocol. Reactions were performed in 25 μl using thefollowing parameters: 2 min. at 50° C.; 10 min. at 95° C.; 15 sec. at95° C./1 min. at 60° C. (40 cycles). Results were recorded as CT values(cycle at which a given sample crosses a threshold level offluorescence) using a log scale, with the difference in RNAconcentration between a given sample and the sample with the lowest CTvalue being represented as 2 to the power of delta CT. The percentrelative expression is then obtained by taking the reciprocal of thisRNA difference and multiplying by 100. The average CT values obtainedfor -actin and GAPDH were used to normalize RNA samples. The RNA samplegenerating the highest CT value required no further diluting, while allother samples were diluted relative to this sample according to theirβ-actin /GAPDH average CT values.

[0377] Normalized RNA (5 μl) was converted to cDNA and analyzed viaTAQMAN® using One Step RT-PCR Master Mix Reagents (PE Biosystems; cat.#4309169) and gene-specific primers according to the manufacturer'sinstructions. Probes and primers were designed for each assay accordingto Perkin Elmer Biosystem's Primer Express Software package (version Ifor Apple Computer's Macintosh Power PC) using the sequence of clone10326230.0.38 as input. Default settings were used for reactionconditions and the following parameters were set before selectingprimers: primer concentration=250 nM, primer melting temperature (T_(m))range=58°-60° C., primer optimal Tm=59° C., maximum primer difference=2°C., probe does not have 5′ G, probe T_(m) must be 10° C. greater thanprimer T_(m), amplicon size 75 bp to 100 bp. The probes and primersselected (see below) were synthesized by Synthegen (Houston, Tex., USA).Probes were double purified by HPLC to remove uncoupled dye andevaluated by mass spectroscopy to verify coupling of reporter andquencher dyes to the 5′ and 3′ ends of the probe, respectively. Theirfinal concentrations were: forward and reverse primers, 900 nM each, andprobe, 200 nM.

[0378] For PCR, normalized RNA from each tissue and each cell line wasspotted in each well of a 96 well PCR plate (Perkin Elmer Biosystems).PCR cocktails including two probes (one specific for FGF-CX and a secondgene-specific probe to serve as an internal standard) were set up using1× TaqMan™ PCR Master Mix for the PE Biosystems 7700, with 5 mM MgCl₂,dNTPs (dA, G, C, U at 1:1:1:2 ratios), 0.25 U/ml AmpliTaq Gold™ (PEBiosystems), and 0.4 U/μl RNase inhibitor, and 0.25 U/μl reversetranscriptase. Reverse transcription was performed at 48° C. for 30minutes followed by amplification/PCR cycles as follows: 95° C. 10 min,then 40 cycles of 95° C. for 15 seconds, 60° C. for 1 minute. TABLE 14Tissue Samples used in TaqMan Expression Analysis. No. Tissue Sample No.Tissue Sample 1 Endothelial cells 49 Renal Ca. 786-0 2 Endothelial cells(treated) 50 Renal Ca. A498 3 Pancreas 51 Renal ca. RXF 393 4 Pancreaticca. CAPAN 2 52 Renal Ca. ACHN 5 Adipose 53 Renal ca. UO-3 I 6 Adrenalgland 54 Renal ca. TK- 10 7 Thyroid 55 Liver 8 Salivary gland 56 Liver(fetal) 9 Pituitary gland 57 Liver Ca. (hepatoblast) HepG2 10 Brain(fetal) 58 Lung 11 Brain (whole) 59 Lung (fetal) 12 Brain (amygdala) 60Lung ca. (small cell) LX-1 13 Brain (cerebellum) 61 Lung Ca. (smallcell) NCI-H69 14 Brain (hippocampus) 62 Lung Ca. (s.cell var.) SHP-77 15Brain (hypothalamus) 63 Lung Ca. (large cell)NCI-H460 16 Brain(substantia nigra) 64 Lung Ca. (non-sm. cell) A549 17 Brain (thalamus)65 Lung Ca. (non-s.cell) NCI-H23 18 Spinal cord 66 Lung ca (non-s.cell)HOP-62 19 CNS Ca. (glio/astro) U87-MG 67 Lung Ca. (non-s.d) NCI-H522 20CNS Ca. (glio/astro) U-118-MG 68 Lung ca. (squam.) SW 900 21 CNS Ca.(astro) SW1783 69 Lung Ca. (squam.) NCI-H596 22 CNS ca.* (neuro; met)SK-N-AS 70 Mammary gland 23 CNS ca. (astro) SF-539 71 Breast Ca.* (pl.effusion) MCF-7 24 CNS ca. (astro) SNB-75 72 Breast ca.* (pl.ef)MDA-MB-231 25 CNS ca. (glio) SNB-19 73 Breast ca.* (pl. effusion) T47D26 CNS ca. (glio) U251 74 Breast ca. BT-549 27 CNS ca. (glio) SF-295 75Breast ca. MDA-N 28 Heart 76 Ovary 29 Skeletal muscle 77 Ovarian ca.OVCAR-3 30 Bone marrow 78 Ovarian ca. OVCAR-4 31 Thymus 79 Ovarian ca.OVCAR-5 32 Spleen 80 Ovarian Ca. OVCAR-8 33 Lymph node 81 Ovarian ca.IGROV-1 34 Colon (ascending) 82 Ovarian ca.* (ascites) SK-OV-3 35Stomach 83 Myometrium 36 Small intestine 84 Uterus 37 Colon Ca. SW480 85Placenta 38 Colon ca.* (SW480 met)SW620 86 Prostate 39 Colon Ca. HT29 87Prostate ca.* (bone met) PC-3 40 Colon Ca. HCT-1 16 88 Testis 41 ColonCa. CaCo-2 89 Melanoma Hs688(A).T 42 Colon Ca. HCT-15 90 Melanoma* (met)Hs688(B).T 43 Colon ca. HCC-2998 91 Melanoma UACC-62 44 Gastric ca.*(liver met) NCI-N87 92 Melanoma M14 45 Bladder 93 Melanoma LOX JMVI 46Trachea 94 Melanoma* (met) SK-MEL-5 47 Kidney 95 Melanoma SK-MEL-28 48Kidney (fetal) 96 Melanoma UACC-257

[0379] The Fibroblast Growth Factor-20-like gene disclosed in thisinvention is expressed in at least the following tissues: MammalianTissue, Colon, Lung, Brain, Liver, Kidney, and Stomach. Expressioninformation was derived from the tissue sources of the sequences thatwere included in the derivation of the sequence of CuraGen Acc. No.CG53135-02.

[0380] The following primers and probe were designed. Each possesses aminimum of three mismatches for corresponding regions of the highlyhomologous human FGF-9 and FGF-16 genes so as to be specific for FGF-CX.Set Ag81b covers the region from base 270 to base 343 of Table 1 (SEQ IDNO: 1). It should not detect other known FGF family members. The primersand probe utilized were: Ag81b (F): (SEQ ID NO:18)5′-GGACCACAGCCTCTTCGGTA-3′; Ag81b (R): (SEQ ID NO:19)5′-TGTCCACACCTCTAATACTGACCAG-3′; and Ag81b (P): (SEQ ID NO:20)5′-FAM-CCCACTGCCACACTGATGAATTCCAA-TAMRA-3′.

[0381] The results from a representative experiment are shown in FIG. 4,Panels A and B. Expression is plotted as a percentage of the sampleexhibiting the highest level of expression. Four replicate runs weremade, presented in variously shaded bars. In 39 normal human tissuesexamined, FGF-CX was found to be most highly expressed in the brain,particularly the cerebellum (FIG. 4, Panels A and B). Other tissues ofthe central nervous system expressed much lower levels of FGF-CX. Of the54 human tumor cell lines examined, FGF-CX was found to be most highlyexpressed in a lung carcinoma cell line (LX-1), a colon carcinoma cellline (SW-480) a colon cancer cell line and metastasis (SW480) and agastric carcinoma cell line (NCI-N87; see FIG. 4, Panels A and B).

[0382] Additional real time expression analysis was done on an extensivepanel of tumor tissues obtained during surgery. These tissues includeportions obtained from the actual tumors themselves, as well as theportions termed “normal adjacent tissue (NAT)”, which typically arealready inflamed and show histological evidence of dysplasia. Aprimer-probe set (Ag81) selected to be specific for FGF-CX was employedin a TaqMan experiment with such surgical tissue samples, in which tworeplicate runs were performed: Ag81 (F): 5′-AGGCAGAAGCGGGAGATAGAT-3′;(SEQ ID NO:21) Ag81 (R): 5′-AGCAGCTTTACCTCATTCACAATG-3′; (SEQ ID NO:22)and Ag81 (P): TET-5′-CCATCTACATCCACCACCAGTTGCAGAA-3′-TAMRA. (SEQ IDNO:23)

[0383] Set Ag81covers the region from base 477 to base 554 of Table 1(SEQ ID NO: 1). The replicates are shown as bars of grey and blackshading in FIG. 4, Panels C and D. The results show dramatically thatfor many matched pairs of tumors and their dysplastic NAT samples,FGF-CX is highly expressed in the NAT but not in the tumor itself; morespecifically, in the parenchymal cells adjacent to the tumor. Examplesin which this matched pattern arises include ovarian cancer, bladdercancer, uterine cancer, lung cancer, prostate cancer and liver cancer.

[0384] Without being limited by theory, it is believed from the resultsin FIG. 4, Panels C and D that FGF-CX may contribute to tumorprogression by paracrine stimulation of the tumor epithelium and/orother components in the host tissue (endothelial cells, stromalfibroblasts, infiltrating lymphocytes, and similar cell types).Likewise, FGF-CX may function to stimulate the components in the hosttissue that synthesize or secrete FGF-CX in an autocrine manner. Thesehost component cells may subsequently act on the tumor compartment.

[0385] The elevated expression profile of FGF-CX relative to unmatchednormal tissue suggests that it plays a prospective or promoting role intumor progression. Therefore, therapeutic targeting of FGF-CX using anyof a number of targeting approaches (including, by way of nonlimitingexample, monoclonal antibodies, ribozymes, antisense oligonucleotides,peptides that neutralize the interaction of FGF-CX with cognatereceptor(s), and small drugs that modulate the unidentified receptor forFGF-CX) is anticipated to have a positive therapeutic impact on diseaseprogression. Likewise, the use of such agents to modulate thebioactivity of FGF-CX in tumor progression is anticipated to synergizeor enhance conventional chemotherapy and radiotherapy. Specific diseaseindications where therapeutic targeting of FGF-CX might be appliedinclude adenocarcinomas of the colon, prostate, lung, kidney, uterus,breast, bladder, ovary.

Example 9 Stimulation of Bromodeoxyuridine Incorporation by RecombinantFGF-CX

[0386] 293-EBNA cells (Invitrogen) were transfected using Lipofectamine2000 according to the manufacturer's protocol (Life Technologies,Gaithersburg, Md.). Cells were supplemented with 10% fetal bovine serum(FBS; Life Technologies) 5 hr post-transfection. To generate protein forBrdU and growth assays (Example 10), cells were washed and fed withDulbecco's modified Eagle medium (DMEM; Life Technologies) 18 hrpost-transfection. After 48 hr, the media was discarded and the cellmonolayer was incubated with 100 μM suramin (Sigma, St. Louis, Mo.) in0.5 ml DMEM for 30 min at 4° C. The suramin-extracted conditioned mediawas then removed, clarified by centrifugation (5 min; 2000×g), andsubjected to TALON metal affinity chromatography according to themanufacturer's instructions (Clontech, Palo Alto, Calif.) takingadvantage of the carboxy-terminal polyhistidine tag. Retained fusionprotein was released by washing the column with imidazole.

[0387] FGF-CX protein concentrations were estimated by Western analysisusing a standard curve generated with a V5-tagged protein of knownconcentration. For Western analysis, conditioned media was harvested 48hr post transfection, and the cell monolayer was then incubated with 0.5ml DMEM containing 100 μM suramin for 30 min at 4° C. Thesuramin-containing conditioned media was then harvested.

[0388] To generate control protein, 293-EBNA cells were transfected withpCEP4 plasmid (Invitrogen) and subjected to the purification procedureoutlined above.

[0389] Recombinant FGF-CX was tested for its ability to induce DNAsynthesis in a bromodeoxyuridine (BrdU) incorporation assay. NIH 3T3cells (ATCC number CRL-1658, American Type Culture Collection, Manassas,Va.), CCD-1070Sk cells (ATCC Number CRL-2091) or MG-63 cells (ATCCNumber CRL-1427) were cultured in 96-well plates to ˜100% confluence,washed with DMEM, and serum-starved in DMEM for 24 hr (NIH 3T3) or 48 hr(CCD-1070Sk and MG-63). Recombinant FGF-CX or control protein was thenadded to the cells for 18 hr. The BrdU assay was performed according tothe manufacturer's specifications (Roche Molecular Biochemicals,Indianapolis, Ind.) using a 5 hr BrdU incorporation time.

[0390] It was found that FGF-CX induced DNA synthesis in NIH 3T3 mousefibroblasts at a half maximal concentration of ˜5 ng/ml (FIG. 5 PanelA). In contrast, protein purified from cells transfected with controlvector did not induce DNA synthesis. It was also found that FGF-CXinduces DNA synthesis, as determined by BrdU incorporation, atcomparable dosing levels in a variety of human cell lines includingCCD-1070Sk normal human skin fibroblasts (FIG. 5, Panel B), CCD-1106keratinocytes (FIG. 5, Panel C), MG-63 osteosarcoma cells (data notshown), and breast epithelial cells.

Example 10 Induction of Cell Proliferation by Recombinant FGF-CX

[0391] To determine if recombinant FGF-CX induces cell proliferation,NIH 3T3 cells were cultured in 6-well plates to ˜50% confluence, washedwith DMEM, and fed with DMEM containing recombinant FGF-CX or controlprotein for 48 hr, and then counted. Cell numbers were determined bytrypsinizing the cells and counting them with a Beckman Coulter Z1series counter (Beckman Coulter, Fullerton, Calif.). It was found thatFGF-CX induces about a 3-fold increase in cell number relative tocontrol protein in this assay (FIG. 6).

[0392] To document morphological changes incident upon proliferation,NIH 3T3 cells were treated for 48 hr with recombinant FGF-CX or controlprotein in DMEM/2% calf serum and photographed with a Zeiss Axiovert 100microscope (Carl Zeiss, Inc., Thornwood, N.Y.).

[0393] In addition to reaching a higher cell density (FIG. 6), NIH 3T3cells cultured in the presence of FGF-CX prepared as described inExample 9 exhibited a disorganized pattern of growth, indicating a lossof contact inhibition (FIG. 7). Furthermore, individual cells were foundto be spindly and refractile. These results show that FGF-CX acts as agrowth factor and suggest that recombinant FGF-CX mediates themorphological transformation of NIH 3T3 cells.

Example 11 Tumor Formation by Ectopic FGF-CX-Transfected NIH 3T3 Cellsin Nude Mice

[0394] NIH 3T3 cells were transfected with pCEP4/Sec-FGF-CX or controlvector using Lipofectamine Plus according to the manufacturer's protocol(Life Technologies). Cells were supplemented with 10% calf serum (CS;Life Technologies) 5 hr post-transfection. It was found thatpCEP4/Sec-FGF-CX-transfected cells were morphologically transformed by48 hr after transfection, and remained so after 2 weeks of selection inhygromycin-containing growth media. In contrast, cells transfected withcontrol vector retained their normal morphology (data not shown). Thusthe transfected cells behave as expected based, for example, on theexperiments reported in Example 10.

[0395] In order to study the induction of ectopic tumors, NIH 3T3 cellswere transfected with various experimental and control vectors. Two daysafter transfection, cells were placed into either DMEM/5% CS (forpFGF-CX-transfected cells) or DMEM/10% CS supplemented with 500 μg/mlhygromycin B (for pCEP4/Sec-FGF-CX-transfected cells). After 2 weeks ofculture, subconfluent cells were trypsinized, neutralized with DMEM/10%CS, washed with PBS and counted. One million cells in PBS were injectedinto the lateral subcutis of female athymic nude mice (JacksonLaboratories, Bar Harbor, Me.).

[0396] NIH 3T3 cells were transfected with FGF-CX expression plasmids(pFGF-CX and pIgκ-FGF-CX) or their appropriate control vectors. We foundthat cells transfected with either of the FGF-CX expression vectors weremorphologically transformed by 48 hr after transfection (data notshown), and possessed a phenotype similar to that generated followingexposure of NIH 3T3 cells to recombinant FGF-CX (FIG. 6). In contrast,cells transfected with control vector retained their normal morphology(data not shown).

[0397] To determine if ectopic expression of FGF-CX in vivo induces thetumorigenicity of NIH 3T3 cells, stable transfectants were generated andinjected subcutaneously into nude mice. By 11 days, all of the animalsinjected with either pFGF-CX or pIgκ-FGF-CX-transfected cells possessedrapidly growing tumors increasing in size by 14 days, whereas none ofthe animals injected with control cells developed tumors by 2 weeks(FIG. 8). Photographs of one mouse receiving control treatment andanother mouse that received cells transfected with an FGF-CX-bearingvector are shown in FIG. 9. These results show that cells transformed bytransfection with vectors harboring the FGF-CX gene promote thedevelopment and growth of tumors in vivo.

Example 12 Expression of FGF-CX

[0398] FGF-CX was expressed essentially as described in Example 6. Theprotein was purified using Ni²⁺-affinity chromatography, subjected toSDS-PAGE under both reducing and nonreducing conditions, and stainedusing Coomassie Blue. The results are shown in FIG. 10. It is seen thatunder both sets of conditions, the protein migrates with an apparentmolecular weight of approximately 29-30 kDa.

Example 13 Stimulation of Bromodeoxyuridine Incorporation by RecombinantFGF-CX

[0399] A dose response experiment for incorporation of BrdU was carriedout using human renal carcinoma cells (786-0; American Type CultureCollection, Manassas, Va.). The results are shown in FIG. 11, in whichFGF-CX is designated “20858”. It is seen that FGF-CX stimulatesproliferation of renal carcinoma cells by more than 4-fold overcontrols, with a half-effective dose being about 2.5 ng/mL.

Example 14 Formation of in vitro Foci in Cells Transfected with FGF-CX

[0400] To assess the effect of ectopic FGF-CX expression on cell growthin culture, NIH 3T3 cells were transfected with FGF-CX expressionplasmids (identified as pFGF-20 and pIgκ-FGF-20 in FIG. 12, see Example7) or control vector. NIH 3T3 cells were transfected usingLipofectamine-Plus according to the manufacturer's protocol (LifeTechnologies). Cells were supplemented with 10% calf serum (CS; LifeTechnologies) 5 h post-transfection. Two days after transfection, cellswere transferred to 90-mm dishes and cultured for two weeks in DMEM+5%calf serum. The cells were then stained with a 0.2% crystal violet/70%ethanol solution and photographed. Each 90 mm dish represents half ofthe cells from a 35 mm dish that had been transfected with 1.5 ug ofplasmid DNA.

[0401] It was found that cells transfected with either of the two FGF-CXexpression vectors generated foci of morphologically transformed cellsapproximately 2 weeks after transfection, while cells transfected withcontrol vector retained their normal morphology (FIG. 12). ThepIgκ-FGF-20 construct proved to be significantly more efficient atformation of foci, which are small in the image shown due toovercrowding, than the pFGF-20 construct (see FIG. 12).

Example 15 Receptor Binding Specificity of FGF-CX

[0402] Fibroblast growth factors (FGFs) play important roles in diversefunctions including morphogenesis, cellular differentiation,angiogenesis, tissue remodeling, inflammation, and oncogenesis. FGFscontain a conserved 120-amino acid FGF core domain with a commontertiary structure. FGF signaling is generally assumed to occur byactivation of transmembrane tyrosine kinase receptors. Four FGFreceptors, FGFR1 through FGFR4, have been identified, and activating orinactivating receptor mutations have been described for a subset ofthese genes in both mice and humans.

[0403] To determine the receptor binding specificity of FGF-CX, weexamined the effect of soluble FGF receptors (FGFRs) on the induction ofDNA synthesis in NIH 3T3 cells by recombinant FGF-CX. Four receptorshave been identified to date (Klint P and Claesson-Welsh L. Front.Biosci., 4: 165-177, 1999; Xu X, et al. Cell Tissue Res., 296: 33-43,1999). Soluble receptors for FGFR1β(IIIc), FGFR2α(IIIb), FGFR2β(IIIb),FGFR2α(IIIc), FGFR3α(IIIc) and FGFR4 were utilized. It was found thatsoluble forms of each of these FGFRs were able to specifically inhibitthe biological activity of FGF-CX (see FIG. 13). Complete or nearlycomplete inhibition was obtained with soluble FGFR2α(IIIb),FGFR2β(IIIb), FGFR2α(IIIc), and FGFR3α(IIIc), whereas partial inhibitionwas achieved with soluble FGFR1β(IIIc) and FGFR4. None of the solublereceptor reagents interfered with the induction of DNA synthesis byPDGF-BB, thereby demonstrating their specificity. The integrity of eachsoluble receptor reagent was demonstrated by showing its ability toinhibit the induction of DNA synthesis by aFGF (acidic FGF), a factorknown to interact with all of the FGFRs under analysis.

Example 16 Cloning and Expression of an N-terminal Deletion Form ofFGF-CX

[0404]E. coli strain BL21 (DE3) (Invitrogen) harboring the plasmidpET24a- FGF20X-del54-codon were grown in LB medium at 37° C. Thisplasmid encodes the C-terminal portion of FGF-CX beginning at position55. When cell densities reached an OD of 0.6, IPTG was added to finalconcentration of 1 mM. Induced cultures were then incubated for anadditional 4 hours at 37° C. Cells were harvested by centrifugation at3000×g for 15 minutes at 4° C., suspended in PBS and then disrupted withtwo passes through a microfluidizer. To separate soluble and insolubleproteins, the lysate was subjected to centrifugation at 10,000×g for 20minutes at 4° C. The insoluble fraction (pellet) was extracted with PBScontaining 1M L-arginine. The remaining insoluble material was thenremoved by centrifugation and the soluble fraction of the arginineextract was filtered through 0.2 micron low-protein binding membrane andanalyzed by SDS PAGE. The result is shown in FIG. 14, which indicatesthat the product is a polypeptide with an apparent molecular weight ofapproximately 20 kDa (see arrow). N-terminal sequencing of the expressedpolypeptide provides the sequence AQLAHLHGILRRRQL which is 100%identical to residues 55-69 of FGF-CX (Table 1, SEQ ID NO: 2).

Example 17 Stimulation of Bromodeoxyuridine Incorporation into NIH 3T3Cells in Response to a Truncated Form of FGF-CX

[0405] A vector expressing residues 24-211 of FGF-CX ((d1-23)FGF-CX; SeeTable 1 and SEQ ID NO: 2) was prepared. The incorporation of BrdU by NIH3T3 cells treated with conditioned medium obtained using the vectorincorporating this truncated form was compared to the incorporation inresponse to treatment with conditioned medium using a vector encodingfull length FGF-CX. This experiment was carried out as described inExample 9.

[0406] The results are shown in FIG. 15. It is seen that (d1-23)FGF-CXretains high activity at the lowest concentration tested, 10 ng/mL. Atthis concentration, the activity of full length FGF-CX has fallenconsiderably, approaching the level of the control. It is estimated that(d1-23)FGF-CX may be at least 5-fold more active than full lengthFGF-CX.

Example 18 Cloning and Expression of FGF-CX Variant CG53135-02

[0407] A nucleotide sequence encoding a variant of Fibroblast GrowthFactor-20-like protein, referred to as Acc. No. CG53135-02, wasidentified. The sequence of Acc. No. CG53135-02 was derived bylaboratory cloning of cDNA fragments, by in silico prediction of thesequence. cDNA fragments covering either the full length of the DNAsequence, or part of the sequence, or both, were cloned. In silicoprediction was based on sequences available in CuraGen's proprietarysequence databases or in the public human sequence databases, andprovided either the full length DNA sequence, or some portion thereof.The laboratory cloning was performed using one or more of the methodssummarized below:

[0408] SeqCalling™ Technology: cDNA was derived from various humansamples representing multiple tissue types, normal and diseased states,physiological states, and developmental states from different donors.Samples were obtained as whole tissue, primary cells or tissue culturedprimary cells or cell lines. Cells and cell lines may have been treatedwith biological or chemical agents that regulate gene expression, forexample, growth factors, chemokines or steroids. The cDNA thus derivedwas then sequenced using CuraGen's proprietary SeqCalling technology.Sequence traces were evaluated manually and edited for corrections ifappropriate. cDNA sequences from all samples were assembled together,sometimes including public human sequences, using bioinformatic programsto produce a consensus sequence for each assembly. Each assembly isincluded in CuraGen Corporation's database. Sequences were included ascomponents for assembly when the extent of identity with anothercomponent was at least 95% over 50 bp. Each assembly represents a geneor Hi portion thereof and includes information on variants, such assplice forms single nucleotide polymorphisms (SNPs), insertions,deletions and other sequence variations.

[0409] Exon Linking: The cDNA coding for the CG53135-02 sequence wascloned by the polymerase chain reaction (PCR) using the primers:5′-AGGTCACCATGGCTGTTATTGGC-3′ (SEQ ID NO: 26) and5′-CTGTCTGTCCTCAGAAGAAGTTCTTGATC-3′(SEQ ID NO: 27). Primers weredesigned based on in silico predictions of the full length or someportion (one or more exons) of the cDNA/protein sequence of theinvention. These primers were used to amplify a cDNA from a poolcontaining expressed human sequences derived from the following tissues:adrenal gland, bone marrow, brain-amygdala, brain-cerebellum,brain-hippocampus, brain-substantia nigra, brain-thalamus, brain-whole,fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney,lymphoma-Raji, mammary gland, pancreas, pituitary gland, placenta,prostate, salivary gland, skeletal muscle, small intestine, spinal cord,spleen, stomach, testis, thyroid, trachea and uterus.

[0410] Multiple clones were sequenced and these fragments were assembledtogether, sometimes including public human sequences, usingbioinformatic programs to produce a consensus sequence for eachassembly. Each assembly is included in CuraGen Corporation's database.Sequences were included as components for assembly when the extent ofidentity with another component was at least 95% over 50 bp. Eachassembly represents a gene or portion thereof and includes informationon variants, such as splice forms single nucleotide polymorphisms(SNPs), insertions, deletions and other sequence variations.

[0411] Physical clone: The PCR product derived by exon linking, coveringthe entire open reading frame, was cloned into the pCR2.1 vector fromInvitrogen to provide clone 137627::160083874.1043010.A9.

[0412] The DNA sequence and protein sequence for a novel FibroblastGrowth Factor-20-like gene were obtained by exon linking and arereported here as CuraGen Acc. No. CG53135-02.

[0413] The novel nucleic acid (SEQ ID NO: 28) of 540 nucleotides(designated CuraGen Acc. No. CG53135-02) encoding a novel FibroblastGrowth Factor-20-like protein (SEQ ID NO: 29) is shown in Table 15. Anopen reading frame was identified beginning at nucleotides 1-3 andending at nucleotides 538-540. This polypeptide represents a novelfunctional Fibroblast Growth Factor-20-like protein. The start and stopcodons of the open reading frame are highlighted in bold type. Putativeuntranslated regions (underlined), if any, are found upstream from theinitiation codon and downstream from the termination codon. The encodedprotein having 179 amino acid residues is presented using the one-lettercode in Table 16. TABLE 15 Nucleotide sequence (SEQ ID NO:28) encodingthe Fibroblast Growth Factor-20-like protein of theinvention. >CG53135-02ATGGCTCCCTTAGCCGAAGTCGGGGGCTTTCTGGGCGGCCTGGAGGGCTTGGGCCAGCCG  60GGGGCAGCGCAGCTGGCGCACCTGCACGGCATCCTGCGCCGCCGGCAGCTCTATTGCCGC 120ACCGGCTTCCACCTGCAGATCCTGCCCGACGGCAGCGCGCAGGGCACCCGGCAGGACCAC 180AGCCTCTTCGGTATCTTGGAATTCATCAGTGTGGCAGTGGGACTGGTCAGTATTAGAGGT 240GTGGACAGTGGTCTCTATCTTGGAATGAATGACAAAGGAGAACTCTATGGATCAGAGAAA 300CTTACTTCCGAATGCATCTTTAGGGAGCAGTTTGAAGAGAACTGGTATAACACCTATTCA 360TCTAACATATATAAACATGGAGACACTGGCCGCAGGTATTTTGTGGCACTTAACAAAGAC 420GGAACTCCAAGAGATGGCGCCAGGTCCAAGAGGCATCAGAAATTTACACATTTCTTACCT 480AGACCAGTGGATCCAGAAAGAGTTCCAGAATTGTACAAGGACCTACTGATGTACACTTAG 540

[0414] TABLE 16 Protein sequence (SEQ ID NO:29) encoded by thenucleotide sequence shown in Table 15 above. >CG53135-02MAPLAEVGGFLGGLEGLGQPGAAQLAHLHGILRRRQLYCRTGFHLQILPDGSAQGTRQDHSLFGILEFISVAVGLVSIRGVDSGLYLGMNDKGELYGSEKLTSECIFREQFEENWYNTYSSNIYKHGDTGRRYFVALNKDGTPRDGARSKRHQKFTHFLPRPVDPERVPELYKDLLMYT

[0415] In a search of sequence databases, it was found, for example,that the nucleic acid sequence of this invention has 495 of 506 bases(97%) identical to a gb:GENBANK-ID;AB044277|jacc:AB044277.1 mRNA fromHomo sapiens (Homo sapiens mRNA for FGF-20, complete cds) (Table 10).The full amino acid sequence of the protein of the invention was foundto have 160 of 162 amino acid residues (98%) identical to, and 160 of162 amino acid residues (98%) similar to, the 211 amino acid residueptnr:SWISSNEW-ACC:Q9NP95 protein from Homo sapiens (Human) (FIBROBLASTGROWTH FACTOR-20 (FGF-20))(Table 10).

[0416] A multiple sequence alignment is given in Table 17, with theprotein of the invention being shown on the first line in a ClustalWanalysis comparing the protein of the invention with related proteinsequences. Note this sequence represents a splice form of FibroblastGrowth Factor-20 as indicated in positions 20-51.

[0417] In the alignment shown in Table 17, black outlined amino acidresidues indicate residues identically conserved between sequences(i.e., residues that may be required to preserve structural orfunctional properties); amino acid residues with a gray background aresimilar to one another between sequences, possessing comparable physicaland/or chemical properties without altering protein structure orfunction (e.g. the group L, V, I, and M may be considered similar); andamino acid residues with a white background are neither conserved norsimilar between sequences.

[0418] The presence of identifiable domains in the protein disclosedherein was determined by searches versus domain databases such as Pfam,PROSITE, ProDom, Blocks or Prints and then identified by the Interprodomain accession number. Significant match was found to the IPR002209;(HBGF_FGF) domain, as summarized in Table 18. TABLE 18 Domain analysisfor CG53135-02Model    Domain   seq-f    seq-t    hmm-f    hmm-t      score    E-value---------------     -----    -----     -----       -----        -----    -------FGF        1/1         31      162 ..       1        146[]   280.8    2e−81

[0419] IPR002209; (HBGF_FGF) Heparin-binding growth factors I and II(HBGF) (also known as acidic and basic fibroblast growth factors (FGF)are structurally related mitogens which stimulate growth ordifferentiation of a wide variety of cells of mesodermal orneuroectodermal origin. See, e.g., Burgess & Maciag, 1989) Annu. Rev.Biochem. 58: 575-606; Thomas 1988 Trends Biochem. Sci. 13: 327-328.These two proteins belong to a family of growth factors and oncogeneswhich is a member of a superfamily that also contains the interleukin-1proteins, Kunitz-type soybean trypsin inhibitors (STI) andhistactophilin. All have very similar structures, but although the HBGFand interleukin-1 families share some sequence similarity (about 25%),they show none at all to the STIs. See, e.g., Burgess & Maciag, 1989)Annu. Rev. Biochem. 58: 575-606; Thomas 1988 Trends Biochem. Sci. 13:327-328; Heath et al. 1995 Curr. Biol. 5: 500-507; Matthews et al. 1991Proc. Natl. Acad. Sci. U.S.A. 88: 3441-3445; Murzin 1992 J. Mol. Biol.223: 531-543; Gimenez-Gallego et al. 1985 Science 230: 1385-1388;Copeland et al. 1996 Proc. Natl. Acad. Sci. U.S.A. 93: 9850-9857; andAyres et al. 1994 Virology 202: 586-605.

[0420] HBGFs are involved in many different processes related to celldifferentiation and growth control. See, e.g., Burgess & Maciag, 1989)Annu. Rev. Biochem. 58: 575-606. HBGF1 and HBGF2 have similar effects:they induce mesoderm formation in embryogenesis, and mediate woundrepair, angiogenesis and neural outgrowth; they also induceproliferation and migration of fibroblasts, endothelial cells andastroglial cells. HBGF3 (int-2) and HBGF4 (hst/ks) are known oncogenes,from stomach tumors and Kaposi sarcoma respectively. HBGF5 and HBGF6 arealso oncogene products. HBGF7, keratinocyte growth factor, is possiblythe major paracrine effector of normal epithelial cell proliferation.

[0421] These growth factors cause dimerization of their tyrosine kinasereceptors leading to intracellular signaling. There are currently fourknown tyrosine kinase receptors for fibroblast growth factors. Thesereceptors can each bind several different members of this family. See,e.g., Heath et al. 1995 Curr. Biol. 5: 500-507.

[0422] The crystal structures of HBGF1 and HBGF2 have been solved. See,e.g., Matthews et al. 1991 Proc. Natl. Acad. Sci. U.S.A. 88: 3441-3445.HBGF1 and HBGF2 have the same twelve-stranded beta-sheet structure asboth interleukin-1 and the Kunitz-type soybean trypsin inhibitors. See,e.g., Murzin 1992 J. Mol. Biol. 223: 531-543. HBGF1 and interleukin-1had been found to be similar, and they were predicted to have similarstructures. See, e.g., Gimenez-Gallego et al. 1985 Science 230:1385-1388. The beta-sheets are arranged in three similar lobes around acentral axis, six strands forming an anti-parallel beta-barrel. Severalregions of HBGF1 have been implicated in receptor binding, notablybeta-strands one through three, and the loop between strands eight andnine. The loop between strands ten and eleven is thought to be involvedin binding heparin.

[0423] This indicates that the sequence of the invention has propertiessimilar to those of other proteins known to contain the HBGF 1-like andHBGF2-like domain(s) and similar to the properties of these domains.

[0424] The nucleic acids and proteins of the invention have applicationsin the diagnosis and/or treatment of various diseases and disorders. Forexample, the compositions of the present invention will have efficacyfor the treatment of patients suffering from: Hirschsprung's disease,Crohn's Disease, appendicitis, inflammatory bowel disease, diverticulardisease, systemic lupus erythematosus, autoimmune disease, asthma,emphysema, scleroderma, allergy, ARDS, Von Hippel-Lindau (VHL) syndrome,cirrhosis, transplantation, hypercalcemia, ulcers, cardiomyopathy,atherosclerosis, hypertension, congenital heart defects, aorticstenosis, atrial septal defect (ASD), atrioventricular (A-V) canaldefect, ductus arteriosus, pulmonary stenosis, subaortic stenosis,ventricular septal defect (VSD), valve diseases, tuberous sclerosis,scleroderma, obesity, diabetes, autoimmune disease, renal arterystenosis, interstitial nephritis, glomerulonephritis, polycystic kidneydisease, systemic lupus erythematosus, renal tubular acidosis, IgAnephropathy, hypercalcemia, Alzheimer's disease, stroke, tuberoussclerosis, hypercalcemia, Parkinson's disease, Huntington's disease,cerebral palsy, epilepsy, Lesch-Nyhan syndrome, multiple sclerosis,ataxia-telangiectasia, leukodystrophies, behavioral disorders,addiction, anxiety, pain, neurodegeneration as well as other diseases,disorders and conditions.

Example 19 Cloning and Expression of FGF-CX Variant CG53135-06

[0425] A nucleotide sequence (SEQ ID NO: 30) encoding a variant ofFibroblast Growth Factor-20-like protein (SEQ ID NO: 31), referred to asAcc. No. CG53135-06, was identified, as shown in Tables 19 and 20.SeqCalling assembly sequences were initially identified by searchingCuraGen Corporation's proprietary Human SeqCalling® database for DNAsequences that translate into proteins with similarity to SNP variant ofFGF20 and/or members of the FGF20 family. One or more SeqCallingassemblies 174203299 were identified as having suitable similarity. SeeTable 11, above. The selected assembly was analyzed further to identifyany open reading frames encoding novel full length proteins as well asnovel splice forms. The resulting DNA sequence and protein sequence fora novel SNP variant of FGF20-like gene are reported here as CuraGen Acc.No. CG53135-06. TABLE 19 Nucleotide sequence (SEQ ID NO:30) encoding theSNP variant of FGR20-like protein (Acc. No. CG53135-06) of theinvention. >CG53135-06ATGGCTCCCTTAGCCGAAGTCGGGGGCTTTCTGGGCGGCCTGGAGGGCTTGGGCCAGCCG  60GGGGCAGCGCAGCTGGCGCACCTGCACGGCATCCTGCGCCGCCGGCAGCTCTATTGCCGC 120ACCGGCTTCCACCTGCAGATCCTGCCCGACGGCAGCGTGCAGGGCACCCGGCAGGACCAC 180AGCCTCTTCGGTATCTTGGAATTCATCAGTGTGGCAGTGGGACTGGTCAGTATTAGAGGT 240GTGGACAGTGGTCTCTATCTTGGAATGAATGACAAAGGAGAACTCTATGGATCAGAGAAA 300CTTACTTCCGAATGCATCTTTAGGGAGCAGTTTGAAGAGAACTGGTATAACACCTATTCA 360TCTAACATATATAAACATGGAGACACTGGCCGCAGGTATTTTGTGGCACTTAACAAAGAC 420GGAACTCCAAGAGATGGCGCCAGGTCCAAGAGGCATCAGAAATTTACACATTTCTTACCT 480AGACCAGTGGATCCAGAAAGAGTTCCAGAATTGTACAAGGACCTACTGATGTACACTTAG 540

[0426] TABLE 20 Protein sequence (SEQ ID NO:31) encoded by thenucleotide sequence shown in Table 19. >CG53135-06MAPLAEVGGFLGGLEGLGQPGAAQLAHLHGILRRRQLYCRTGFHLQILPDGSVQGTRQDHSLFGILEFISVAVGLVSIRGVDSGLYLGMNDKGELYGSEKLTSECIFREQFEENWYNTYSSNIYKHGDTGRRYFVALNKDGTPRDGARSKRHQKFTHFLPRPVDPERVPELYKDLLMYT

[0427] A multiple sequence alignment is given in Table 21, with theprotein of the invention being shown on the first line in a ClustalWanalysis comparing the protein of the invention with related proteinsequences. Note this sequence represents a SNP of FGF20 as indicated inposition 53 of CG53135-06. Additional SNPs are described in Example 21,below.

[0428] The presence of identifiable domains in the protein disclosedherein was determined by searches versus domain databases such as Pfam,PROSITE, ProDom, Blocks or Prints and then identified by the Interprodomain accession number. Significant domains are summarized in Table 22.The IntroPro IPR002209 FGF domain is described above. TABLE 22 Domainanalysis for CG53135-06 Model Description Score E-value N FGF (InterPro)Fibroblast growth 286.8 3.4e-83 1 factor Parsed for domains: seq- seq-hmm- hmm- Model Domain f t f t score E-value FGF 1/1 31 162 .. 1 146 []286.8 3.4e-83

Example 20 Cloning and Characterization of FGF-CX Variants includingOptimized FGF-CX

[0429] Additional FGF-CX variants were cloned as described above.Nucleotide and polypeptide are shown in Tables 23-29. Codon optimizedFGF-CX is shown in Table 25. TABLE 23 Nucleotide and PolypeptideSequence for CG53135-03, Consensus DNA Sequence: >CG53135-03 636 ntATGGCTCCCTTAGCCGAAGTCGGGGGCTTTCTGGGCGGCCTGGAGGGCTTGGGCCAGCAG (SEQ ID NO:33) GTGGGTTCGCATTTCCTGTTGCCTCCTGCCGGGGAGCGGCCGCCGCTGCTGGGCGAGCGCAGGAGCGCGGCGGAGCGGAGCGCGCGCGGCGGGCCGGGGGCTGCGCAGCTGGCGCACCTGCACGGCATCCTGCGCCGCCGGCAGCTCTATTGCCGCACCGGCTTCCACCTGCAGATCCTGCCCGACGGCAGCGTGCAGGGCACCCGGCAGGACCACAGCCTCTTCGGTATCTTGGAATTCATCAGTGTGGCAGTGGGACTGGTCAGTATTAGAGGTGTGGACAGTGGTCTCTATCTTGGAATGAATGACAAAGGAGAACTCTATGGATCAGAGAAACTTACTTCCGAATGCATCTTTAGGGAGCAGTTTGAAGAGAACTGGTATAACACCTATTCATCTAACATATATAAACATGGAGACACTGGCCGCAGGTATTTTGTGGCACTTAACAAAGACGGAACTCCAAGAGATGGCGCCAGGTCCAAGAGGCATCAGAAATTTACACATTTCTTACCTAGACCAGTGGATCCAGAAAGAGTTCCAGAATTGTACAAGGACCTACTGATGTACACTTGA Protein Sequence: ORFStart: >CG53135-03-prot 211 aa 1 ORF Stop: 634 Frame: 1MAPLAEVGGFLGGLEGLGQQVGSHFLLPPAGERPPLLGERRSAAERSARGGPGAAQLAHL (SEQ ID NO:34) HGILRRRQLYCRTGFHLQILPDGSVQGTRQDHSLFGILEFISVAVGLVSIRGVDSGLYLGMNDKGELYGSEKLTSECIFREQFEENWYNTYSSNIYKHGDTGRRYFVALNKDGTPRDGARSKRHQKFTHFLPRPVDPERVPELYKDLLMYT

[0430] TABLE 24 Nucleotide and Polypeptide Sequence forCG53135-04:Consensus DNA Sequence: >CG53135-04 540 ntATGGCTCCCTTAGCCGAAGTCGGGGGCTTTCTGGGCGGCCTGGAGGGCTTGGGCCAGCCG (SEQ ID NO:35) GGGGCAGCGCAGCTGGCGCACCTGCACGGCATCCTGCGCCGCCGGCAGCTCTATTGCCGCACCGGCTTCCACCTGCAGATCCTGCCCGACGGCAGCGCGCAGGGCACCCGGCAGGACCACAGCCTCTTCGGTATCTTGGAATTCATCAGTGTGGCAGTGGGACTGGTCAGTATTAGAGGTGTGGACAGTGGTCTCTATCTTGGAATGAATGACAAAGGAGAACTCTATGGATCAGAGAAACTTACTTCCGAATGCATCTTTAGGGAGCAGTTTGAAGAGAACTGGTATAACACCTATTCATCTAACATATATAAACATGGAGACACTGGCCGCAGGTATTTTGTGGCACTTAACAAAGACGGAACTCCAAGAGATGGCGCCAGGTCCAAGAGGCATCAGAAATTTACACATTTCTTACCTAGACCAGTGGATCCAGAAAGAGTTCCAGAATTGTACAAGGACCTACTGATGTACACTTAG >CG53135-04-prot179 aa MAPLAEVGGFLGGLEGLGQPGAAQLAHLHGILRRRQLYCRTGFHLQILPDGSAQGTRQDH (SEQID NO: 36) SLFGILEFISVAVGLVSIRGVDSGLYLGMNDKGELYGSEKLTSECIFREQFEENWYNTYSSNIYKHGDTGRRYFVALNKDGTPRDGARSKRHQKFTHFLPRPVDPERVPELYKDLLMYT

[0431] TABLE 25 Nucleotide and Polypeptide Sequence for Clone Assembly,FGF-20X, Codon Optimized, CG53135-05 >CG53135-05 636 ntATGGCTCCGCTGGCTGAAGTTGGTGGTTTCCTGGGCGGTCTGGAGGGTCTGGGTCAGCAG (SEQ ID NO:37) GTTGGTTCTCACTTCCTGCTGCCGCCGGCTGGTGAACGTCCGCCACTGCTGGGTGAACGTCGCTCCGCAGCTGAACGCTCCGCTCGTGGTGGCCCGGGTGCTGCTCAGCTGGCTCACCTGCATGGTATCCTGCGTCGCCGTCAGCTGTACTGCCGTACTGGTTTCCACCTGCAGATCCTGCCGGATGGTTCTGTTCAGGGTACCCGTCAGGACCACTCTCTGTTCGGTATCCTGGAATTCATCTCTGTTGCTGTTGGTCTGGTTTCTATCCGTGGTGTTGACTCTGGCCTGTACCTGGGTATGAACGACAAAGGCGAACTGTACGGTTCTGAAAAACTGACCTCTGAATGCATCTTCCGTGAACAGTTTGAAGAGAACTGGTACAACACCTACTCTTCCAACATCTACAAACATGGTGACACCGGCCGTCGCTACTTCGTTGCTCTGAACAAAGACGGTACCCCGCGTGATGGTGCTCGTTCTAAACGTCACCAGAAATTCACCCACTTCCTGCCGCGCCCAGTTGACCCGGAGCGTGTTCCAGAACTGTATAAAGACCTGCTGATGTACACCTAA Protein Sequence: >CG53135-05-prot211 aa MAPLAEVGGFLGGLEGLGQQVGSHFLLPPAGERPPLLGERRSAAERSARGGPGAAQLAHL (SEQID NO: 38) HGILRRRQLYCRTGFHLQILPDGSVQGTRQDHSLFGILEFISVAVGLVSIRGVDSGLYLGMNDKGELYGSEKLTSECIFREQFEENWYNTYSSNIYKHGDTGRRYFVALNKDGTPRDGARSKRHQKFTHFLPRPVDPERVPELYKDLLMYT

[0432] TABLE 26 Nucleotide and Polypeptide Sequence for CG53135-07:CG53135-07 54 nt ATGGCTCCCTTAGCCGAAGTCGGGGGCTTTCTGGGCGGCCTGGAGGGCTTGGGC(SEQ ID NO: 39) Protein Sequence of CG53135-07: CG53135-07-prot 18 aaMAPLAEVGGFLGGLEGLG (SEQ ID NO: 40)

[0433] TABLE 27 Nucelotide and Polypeptide Sequence for CG53135pep2,CG53135-08: CG53135-08 63 ntGAGCGGCCGCCGCTGCTGGGCGAGCGCAGGAGCGCGGCGGAGCGGAGCGCGCGCGGCGGGCCG (SEQ IDNO: 41) CG53135-08-prot 21 aa ERPPLLGERRSAAERSARGGP (SEQ ID NO: 42)

[0434] TABLE 28 Nucleotide and Polypeptide Sequence for CG53135pep3,CG53135-09: CG53135-09 63 ntCGCAGGTATTTTGTGGCACTTAACAAAGACGGAACTCCAAGAGATGGCGCCAGGTCCAAGAGG (SEQ IDNO: 43) CG53135-09-prot 21 aa RRYFVALNKDGTPRDGARSKR (SEQ ID NO: 44)

[0435] TABLE 29 Nucleotide and Polypeptide Sequences for FGF-20,CG53135pep4, CG53135-10: CG53135-10 60 ntCCTAGACCAGTGGATCCAGAAAGAGTTCCAGAATTGTACAAGGACCTACTGATGTACACT (SEQ ID NO:45) CG53135-10-prot 20 aa PRPVDPERVPELYKDLLMYT (SEQ ID NO: 46)

[0436] A ClustalW alignment of FGF-CX variants CG53135-01 throughCG53135-08 (labeled as v-01 through v-04, respectively) is shown inTable 30.

[0437] BLASTP and BLASTN alignments of the codon optimized FGF-CXnucleic acid (SEQ ID NO: 378) and polypeptide (SEQ ID NO: 38) sequencewere performed. Results are shown in Table 31. TABLE 31hz,1/52 !BLASTanalysis of SEQ ID NOs:37 and 38 Four BLAST searches were performed asindicated: I. CuraBLASTN Analysis of 57optimized from the non-redundantGenBank database II. CuraBLASTN Analysis of 57optimized from thenon-redundant GenBank database III. CuraBLASTN Analysis of 57optimizedfrom GENESEQ PatN database IV. CuraBLASTX Analysis of 57optimized fromthe non-redundant GenBank database Results are summarized for eachsearch: Sum High Probability Sequences producing High-scoring SegmentPairs: Score P(N) I. gb:GENBANK-ID:AX250592|acc:AX250592.1 Sequence1from Pat . . . 1830 6.7e−77 gb:GENBANK-ID:AX356109|acc:AX356109.1Sequence1 from Pat . . . 1830 1.3e−76gb:GENBANK-ID:AX128219|acc:AX128219.1 Sequence3 from Pat . . . 18301.4e−76 gb:GENBANK-ID:AX080364|acc:AX080364.1 Sequence1 from Pat . . .1828 1.7e−76 gb:GENBANK-ID:AX356110|acc:AX356110.1 Sequence2 from Pat .. . 1828 1.7e−76 gb:GENBANK-ID:AX128217|acc:AX128217.1 Sequence1 fromPat . . . 1812 8.9e−76 II. gb:GENBANK-ID:AB044277|acc:AB044277.1 Homosapiens mRNA f . . . 1830 8.1e−77 gb:GENBANK-ID:AB030648|acc:AB030648.1Homo sapiens mRNA f . . . 1830 1.3e−76gb:GENBANK-ID:AB020021|acc:AB020021.1 Rattus norvegicus m . . . 18128.4e−76 gb:GENBANK-ID:AB049218|acc:AB049218.1 Mus musculus Fgf20 . . .1794 5.4e−75 gb:GENBANK-ID:AB020858|acc:AB020858.1 Homo sapiens genomi .. . 854 5.0e−67 III. patn:AAA75630 Nucleotide sequence of humanfibroblast gro . . . 1830 1.1e−76 patn:AAS03277 Human cDNA encodingfibroblast growth facto . . . 1830 1.1e−76 patn:AAF62049 Humanfibroblast growth factor-CX (FGF-CX) . . . 1828 1.4e−76 patn:AAS03276Rat cDNA encoding fibroblast growth factor . . . 1812 7.1e−76patn:AAT58530 Nucleotide sequence of mouse FGF9 derived f . . . 11333.2e−45 IV. ptnr:SWISSPROT-ACC:Q9NP95 Fibroblast growth factor-20 . . .+1 1118 3.3e−112 ptnr:SPTREMBL-ACC:Q9EST9 FGF-20- Rattus norvegicus (R .. . +1 1074 1.5e−107 ptnr:SPTREMBL-ACC:Q9ESL9 FIBROBLAST GROWTH FACTOR20- . . . +1 1069 5.2e−107 ptnr:SPTREMBL-ACC:Q9PVY1 XFGF-20- Xenopuslaevis (Afr . . . +1 906 9.7e−90 ptnr:SWISSPROT-ACC:P31371Glia-activating factor precu . . . +1 775 7.4e−76

Example 21 Cloning and Expression of FGF-CX SNP Variants

[0438] SeqCallingTM Technology: cDNA was derived from various humansamples representing multiple tissue types, normal and diseased states,physiological states, and developmental states from different donors.Samples were obtained as whole tissue, cell lines, primary cells ortissue cultured primary cells and cell lines. Cells and cell lines mayhave been treated with biological or chemical agents that regulate geneexpression for example, growth factors, chemokines, steroids. The cDNAthus derived was then sequenced using CuraGen's proprietary SeqCallingtechnology. Sequence traces were evaluated manually and edited forcorrections if appropriate. cDNA sequences from all samples wereassembled with themselves and with public ESTs using bioinformaticsprograms to generate CuraGen's human SeqCalling database of SeqCallingassemblies. Each assembly contains one or more overlapping cDNAsequences derived from one or more human samples. Fragments and ESTswere included as components for an assembly when the extent of identitywith another component of the assembly was at least 95% over 50 bp. Eachassembly can represent a gene and/or its variants such as splice formsand/or single nucleotide polymorphisms (SNPs) and their combinations.

[0439] Variant sequences identified in human genomic DNA are included inthis application. A variant sequence can include a single nucleotidepolymorphism (SNP). A SNP can, in some instances, be referred to as a“cSNP” to denote that the nucleotide sequence containing the SNPoriginates as a cDNA. A SNP can arise in several ways. For example, aSNP may be due to a substitution of one nucleotide for another at thepolymorphic site. Such a substitution can be either a transition or atransversion. A SNP can also arise from a deletion of a nucleotide or aninsertion of a nucleotide, relative to a reference allele. In this case,the polymorphic site is a site at which one allele bears a gap withrespect to a particular nucleotide in another allele. SNPs occurringwithin genes may result in an alteration of the amino acid encoded bythe gene at the position of the SNP. Intragenic SNPs may also be silent,however, in the case that a codon including a SNP encodes the same aminoacid as a result of the redundancy of the genetic code. SNPs occurringoutside the region of a gene, or in an intron within a gene, do notresult in changes in any amino acid sequence of a protein but may resultin altered regulation of the expression pattern for example, alterationin temporal expression, physiological response regulation, cell typeexpression regulation, intensity of expression, stability of transcribedmessage.

[0440] Method of novel SNP Identification: SNPs were identified byanalyzing genomic sequence assemblies generated by a process called DeepSNP Mining (DSM) in CuraGen's proprietary SNPTool algorithm. The SNPToolidentifies variation in assemblies with the following criteria: SNPs arenot analyzed within 10 base pairs on both ends of an alignment; windowsize (number of bases in a view) is 10; the allowed number of mismatchesin a window is 2; minimum SNP base quality (PHRED score) is 23; minimumnumber of changes to score an SNP is 2/assembly position. SNPToolanalyzes the assembly and displays SNP positions, associated individualvariant sequences in the assembly, the depth of the assembly at thatgiven position, the putative assembly allele frequency, SNP sequencevariation, and the genomic DNA pool source. Sequence traces are thenselected and brought into view for manual validation. Built-inFrameSearch software allows for the concurrent identification of aminoacid changing SNPs. SNPs that border the intron/exon boundary weredouble checked by importing the SNP consensus into CuraTools andperforming a 1×1 TBLASTN against the CGUID protein sequence of interest.Comprehensive SNP data analysis is then exported into the SNPCallingdatabase.

[0441] Method of novel SNP Confirmation: SNPs are confirmed employing avalidated method know as Pyrosequencing. Detailed protocols forPyrosequencing can be found in: Alderborn et al.. (2000). GenomeResearch. 10, Issue 8, August. 1249-1265. SNP results are shown in Table32. TABLE 32 Variants of nucleotide sequence described in FIG. 1.Nucleotides Amino Acids Variant Position Initial Modified PositionInitial Modified 13377871 301 A G 101 IIe Val 13374151 308 T G 103 ValGly 13375519 361 A G 121 Met VaJ 13375518 517 G A 173 Gly Arg 13375516523 C G 175 Pro Ala 13375517 616 G A 206 Asp Asn

Example 22 Molecular Cloning of FGF-CX Variant CG53 135-04

[0442] A. Molecular Cloning of CG53135-04 residue 1 to 179

[0443] The cDNA coding for the full-length form of CG53135-04 fromresidue 1 to 179 was targeted for “in-frame” cloning by PCR. The PCRtemplate is based on the previously identified plasmid.

[0444] The following oligonucleotide primers were used to clone thetarget cDNA sequence:

[0445] F1 5′-CACCAGATCT ATGGCTCCCTTAGCCGAAGTCGGGGGC-3′(SEQ ID NO: 55)

[0446] R1 5′-GCCGTCGAC AGTGTACATCAGTAGGTCCTTGTACAATTC-3′(SEQ ID NO: 56)

[0447] For downstream cloning purposes, the forward primer includes anin-frame Bgl II restriction site and the reverse primer contains anin-frame Sal I restriction site.

[0448] Two PCR reactions were set up using a total of 1-5 ng of theplasmid that contains the insert for CG53135-04.

[0449] The reaction mixtures contained 2 microliters of each of theprimers (original concentration: 5 pmol/ul), 1 microliter of 10 mM dNTP(Clontech Laboratories, Palo Alto Calif.) and 1 microliter of50×Advantage-HF 2 polymerase (Clontech Laboratories) in 50microliter-reaction volume. The following reaction conditions were used:

[0450] PCR condition 1:

[0451] a) 96° C. 3 minutes

[0452] b) 96° C. 30 seconds denaturation

[0453] c) 60° C. 30 seconds, primer annealing

[0454] d) 72° C. 6 minutes extension

[0455] Repeat steps b-d 15 times

[0456] e) 96° C. 15 seconds denaturation

[0457] f) 60° C. 30 seconds, primer annealing

[0458] g) 72° C. 6 minutes extension

[0459] Repeat steps e-g 29 times

[0460] e) 72° C. 10 minutes final extension

[0461] PCR condition 2:

[0462] a) 96° C. 3 minutes

[0463] b) 96° C. 15 seconds denaturation

[0464] c) 76° C. 30 seconds, reducing the temperature by 1° C. per cycle

[0465] d) 72° C. 4 minutes extension

[0466] Repeat steps b-d 34 times

[0467] e) 72° C. 10 minutes final extension.

[0468] An amplified product was detected by agarose gel electrophoresis.The fragment was gel-purified and ligated into the pCR2.1 TOPO vector(Invitrogen, Carlsbad, Calif.) following the manufacturer'srecommendation. Twelve clones per PCR reaction were picked andsequenced. The inserts were sequenced using vector-specific M13 Forwardand M13 Reverse primers. SF1: GTATCTTGGAATTCATCAGTGTGGC (SEQ ID NO:57)SF2: TGGTCTCTATCTTGGAATGAATGAC (SEQ ID NO:58) SR1: GAAGAGGCTGTGGTCCTGCC(SEQ ID NO:59) SR2: ACTGTCCACACCTCTAATACTGACC (SEQ ID NO:60)

[0469] The insert assembly 250059596 was found to encode an open readingframe between residues 1 and 179 of the target sequence of CG53135-04.See Tables 33-36. The cloned inserts are 100% identical to the originalsequence. The alignment with CG53135-04 is displayed in a ClustalW inTable 37. The first 3 and the last 3 amino acid residues of theassemblies are derived from the restriction enzyme sites added in theprimers for the purpose of sub-cloning. Note that differing amino acidshave a white or grey background, and deleted/inserted amino acids can bedetected by a dashed line in the sequence that does not code at thatposition. TABLE 33 Cloned Sequences >CG53135-04ATGGCTCCCTTAGCCGAAGTCGGGGGCTTTCTGGGCGGCCTGGAGGGCTTGGGCCAGCCGGGGGCAGCGC(SEQ ID NO:61)AGCTGGCGCACCTGCACGGCATCCTGCGCCGCCGGCAGCTCTATTGCCGCACCGGCTTCCACCTGCAGATCCTGCCCGACGGCAGCGTGCAGGGCACCCGGCAGGACCACAGCCTCTTCGGTATCTTGGAATTCATCAGTGTGGCAGTGGGACTGGTCAGTATTAGAGGTGTGGACAGTGGTCTCTATCTTGGAATGAATGACAAAGGAGAACTCTATGGATCAGAGAAACTTACTTCCGAATGCATCTTTAGGGAGCAGTTTGAAGAGAACTGGTATAACACCTATTCATCTAACATATATAAACATGGAGACACTGGCCGCAGGTATTTTGTGGCACTTAACAAAGACGGAACTCCAAGAGATGGCGCCAGGTCCAAGAGGCATCAGAAATTTACACATTTCTTACCTAGACCAGTGGATCCAGAAAGAGTTCCAGAATTGTACAAGGACCTACTGATGTACACTTAG

[0470] TABLE 34 Cloned Sequences >250059596CACCAGATCTATGGCTCCCTTAGCCGAAGTCGGGGGCTTTCTGGGCGGCCTGGAGGGCTTGGGCCAGCCG(SEQ ID NO:62)GGGGCAGCGCAGCTGGCGCACCTGCACGGCATCCTGCGCCGCCGGCAGCTCTATTGCCGCACCGGCTTCCACCTGCAGATCCTGCCCGACGGCAGCGTGCAGGGCACCCGGCAGGACCACAGCCTCTTCGGTATCTTGGAATTCATCAGTGTGGCAGTGGGACTGGTCAGTATTAGAGGTGTGGACAGTGGTCTCTATCTTGGAATGAATGACAAAGGAGAACTCTATGGATCAGAGAAACTTACTTCCGAATGCATCTTTAGGGAGCAGTTTGAAGAGAACTGGTATAACACCTATTCATCTAACATATATAAACATGGAGACACTGGCCGCAGGTATTTTGTGGCACTTAACAAAGACGGAACTCCAAGAGATGGCGCCAGGTCCAAGAGGCATCAGAAATTTACACATTTCTTACCTAGACCAGTGGATCCAGAAAGAGTTCCAGAATTGTACAAGGACCTACTGATGTACACTGTCGACGGC

[0471] TABLE 35 View DNA Sequence Analysis of CG53135-04 TranslatedProtein — Frame: 1 — Nucleotide 1 to 537 1ATGGCTCCCTTAGCCGAAGTCGGGGGCTTTCTGGGCGGCCTGGAGGGCTTGGGCCAGCCGGGGGCAGCGCAGCTGGCGCAM  A  P  L  A  E  V  G  G  F  L  G  G  L  E  G  L  G  Q  P  G  A  A  Q  L  A  H81CCTGCACGGCATCCTGCGCCGCCGGCAGCTCTATTGCCGCACCGGCTTCCACCTGCAGATCCTGCCCGACGGCAGCGTGC L  H  G  I  L  R  R  R  Q  L  Y  C  R  T  G  F  H  L  Q  I  L  P  D  G  S  V  Q161AGGGCACCCGGCAGGACCACAGCCTCTTCGGTATCTTGGAATTCATCAGTGTGGCAGTGGGACTGGTCAGTATTAGAGGT  G  T  R  Q  D  H  S  L  F  G  I  L  E  F  I  S  V  A  V  G  L  V  S  I  R  G241GTGGACAGTGGTCTCTATCTTGGAATGAATGACAAAGGAGAACTCTATGGATCAGAGAAACTTACTTCCGAATGCATCTTV  D  S  G  L  Y  L  G  M  N  D  K  G  E  L  Y  G  S  E  K  L  T  S  E  C  I  F321TAGGGAGCAGTTTGAAGAGAACTGGTATAACACCTATTCATCTAACATATATAAACATGGAGACACTGGCCGCAGGTATT R  E  Q  F  E  E  N  W  Y  N  T  Y  S  S  N  I  Y  K  E  G  D  T  G  R  R  Y  F401TTGTGGCACTTAACAAAGACGGAACTCCAAGAGATGGCGCCAGGTCCAAGAGGCATCAGAAATTTACACATTTCTTACCT  V  A  L  N  K  D  G  T  P  R  D  G  A  R  S  K  R  H  Q  K  F  T  H  F  L  P481 AGACCAGTGGATCCAGAAAGAGTTCCAGAATTGTACAAGGACCTACTGATGTACACTTAGR  P  V  D  P  E  R  V  P  E  L  Y  K  D  L  L  M  Y  T

[0472] TABLE 36 View DNA Sequence Analysis of 250059596 TranslatedProtein — Frame: 2 — Nucleotide 2 to 556 1CACCAGATCTATGGCTCCCTTAGCCGAAGTCGGGGGCTTTCTGGGCGGCCTGGAGGGCTTGGGCCAGCCGGGGGCAGCGC T  R  S  M  A  P  L  A  E  V  G  G  F  L  G  G  L  E  G  L  G  Q  P  G  A  A  Q81AGCTGGCGCACCTGCACGGCATCCTGCGCCGCCGGCAGCTCTATTGCCGCACCGGCTTCCACCTGCAGATCCTGCCCGAC  L  A  H  L  H  G  I  L  R  R  R  Q  L  Y  C  R  T  G  F  H  L  Q  I  L  P  D161GGCAGCGTGCAGGGCACCCGGCAGGACCACAGCCTCTTCGGTATCTTGGAATTCATCAGTGTGGCAGTGGGACTGGTCAGG  S  V  Q  G  T  R  Q  D  H  S  L  F  G  I  L  E  F  I  S  V  A  V  G  L  V  S241TATTAGAGGTGTGGACAGTGGTCTCTATCTTGGAATGAATGACAAAGGAGAACTCTATGGATCAGAGAAACTTACTTCCG I  R  G  V  D  S  G  L  Y  L  G  M  N  D  K  G  E  L  Y  G  S  E  K  L  T  S E 321AATGCATCTTTAGGGAGCAGTTTGAAGAGAACTGGTATAACACCTATTCATCTAACATATATAAACATGGAGACACTGGC  C  I  F  R  E  Q  F  E  E  N  W  Y  N  T  Y  S  S  N  I  Y  K  H  G  D  T  G401CGCAGGTATTTTGTGGCACTTAACAAAGACGGAACTCCAAGAGATGGCGCCAGGTCCAAGAGGCATCAGAAATTTACACAR  R  Y  F  V  A  L  N  K  D  G  T  P  R  D  G  A  R  S  K  R  H  Q  K  F  T  H481TTTCTTACCTAGACCAGTGGATCCAGAAAGAGTTCCAGAATTGTACAAGGACCTACTGATGTACACTGTCGACGGC F  L  P  R  P  V  D  P  E  R  V  P  E  L  Y  K  D  L  L  M  Y  T  V  D  G

[0473]

[0474] B. MOLECULAR CLONING OF CG53135-04 (31-162 AA)

[0475] The cDNA coding for the domain of CG53 135-04 from residue 31 to162 was targeted for “in-frame” cloning by PCR. The PCR template isbased on the previously identified plasmid.

[0476] The following oligonucleotide primers were used to clone thetarget cDNA sequence: F2 5′-CACCAGATCT ATCCTGCGCCGCCGGCAGCTCTATTGCC-3′(SEQ ID NO:63) R2 5′-GCCGTCGAC TGGTCTAGGTAAGAAATGTGTAAATTTCTGATGCC-3′(SEQ ID NO:64)

[0477] For downstream cloning purposes, the forward primer includes anin-frame Bgl II restriction site and the reverse primer contains anin-frame Sal I restriction site.

[0478] Two PCR reactions were set up using a total of 1 -5 ng of theplasmid that contains the insert for CG53135-04.

[0479] The reaction mixtures contained 2 microliters of each of theprimers (original concentration: 5 pmol/ul), 1 microliter of 10 mM dNTP(Clontech Laboratories, Palo Alto Calif.) and 1 microliter of50×Advantage-HF 2 polymerase (Clontech Laboratories) in 50microliter-reaction volume. The reaction conditions used are provided inabove in Example 22A.

[0480] An amplified product was detected by agarose gel electrophoresis.The fragment was gel-purified and ligated into the pCR2.1 TOPO vector(Invitrogen, Carlsbad, Calif.) following the manufacturer'srecommendation. Twelve clones per PCR reaction were picked andsequenced. The inserts were sequenced using vector-specific M13 Forwardand M13 Reverse primers.

[0481] The insert assembly 250059629 was found to encode an open readingframe between residues 31 and 162 of the target sequence of CG53135-04.The cloned inserts are 100% identical to the original sequence. SeeTables 38-41. The alignment with CG53135-04 is displayed in a ClustalWin Table 42. The first 3 and the last 3 amino acid residues of theassemblies are derived from the restriction enzyme sites added in theprimers for the purpose of sub-cloning. Note that differing amino acidshave a white or grey background, and deleted/inserted amino acids can bedetected by a dashed line in the sequence that does not code at thatposition. TABLE 38 Cloned Sequences >CG53135-04ATGGCTCCCTTAGCCGAAGTCGGGGGCTTTCTGGGCGGCCTGGAGGGCTTGGGCCAGCCGGGGGCAGCGC(SEQ ID NO:65)AGCTGGCGCACCTGCACGGCATCCTGCGCCGCCGGCAGCTCTATTGCCGCACCGGCTTCCACCTGCAGATCCTGCCCGACGGCAGCGTGCAGGGCACCCGGCAGGACCACAGCCTCTTCGGTATCTTGGAATTCATCAGTGTGGCAGTGGGACTGGTCAGTATTAGAGGTGTGGACAGTGGTCTCTATCTTGGAATGAATGACAAAGGAGAACTCTATGGATCAGAGAAACTTACTTCCGAATGCATCTTTAGGGAGCAGTTTGAAGAGAACTGGTATAACACCTATTCATCTAACATATATAAACATGGAGACACTGGCCGCAGGTATTTTGTGGCACTTAACAAAGACGGAACTCCAAGAGATGGCGCCAGGTCCAAGAGGCATCAGAAATTTACACATTTCTTACCTAGACCAGTGGATCCAGAAAGAGTTCCAGAATTGTACAAGGACCTACTGATGTACACTTAG

[0482] Table 39 Cloned Sequences >250059629CACCAGATCTATCCTGCGCCGCCGGCAGCTCTATTGCCGCACCGGCTTCCACCTGCAGATCCTGCCCGAC(SEQ ID NO:66)GGCAGCGTGCAGGGCACCCGGCAGGACCACAGCCTCTTCGGTATCTTGGAATTCATCAGTGTGGCAGTGGGACTGGTCAGTATTAGAGGTGTGGACAGTGGTCTCTATCTTGGAATGAATGACAAAGGAGAACTCTATGGATCAGAGAAACTTACTTCCGAATGCATCTTTAGGGAGCAGTTTGAAGAGAACTGGTATAACACCTATTCATCTAACATATATAAACATGGAGACACTGGCCGCAGGTATTTTGTGGCACTTAACAAAGACGGAACTCCAAGAGATGGCGCCAGGTCCAAGAGGCATCAGAAATTTACACATTTCTTACCTAGACCAGTCGACGGC

[0483] TABLE 40 View DNA Sequence Analysis of CG53135-04 TranslatedProtein - Frame: 1 - Nucleotide 1 to 537 1ATGGCTCCCTTAGCCGAAGTCGGGGGCTTTCTGGGCGGCCTGGAGGGCTTGGGCCAGCCGGGGGCAGCGCAGCTGGCGCAM  A  P  L  A  E  V  G  G  F  L  G  G  L  E  G  L  G  Q  P  G  A  A  Q  L  A  H81CCTGCACGGCATCCTGCGCCGCCGGCAGCTCTATTGCCGCACCGGCTTCCACCTGCAGATCCTGCCCGACGGCAGCGTGC L  H  G  I  L  R  R  R  Q  L  Y  C  R  T  G  F  H  L  Q  I  L  P  D  G  S  V  Q161AGGGCACCCGGCAGGACCACAGCCTCTTCGGTATCTTGGAATTCATCAGTGTGGCAGTGGGACTGGTCAGTATTAGAGGT  G  T  R  Q  D  H  S  L  F  G  I  L  E  F  I  S  V  A  V  G  L  V  S  I  R  G241GTGGACAGTGGTCTCTATCTTGGAATGAATGACAAAGGAGAACTCTATGGATCAGAGAAACTTACTTCCGAATGCATCTTV  D  S  G  L  Y  L  G  M  N  D  K  G  E  L  Y  G  S  E  K  L  T  S  E  C  I  F321TAGGGAGCAGTTTGAAGAGAACTGGTATAACACCTATTCATCTAACATATATAAACATGGAGACACTGGCCGCAGGTATT R  E  Q  F  E  E  N  W  Y  N  T  Y  S  S  N  I  Y  K  H  G  D  T  G  R  R  Y  F401TTGTGGCACTTAACAAAGACGGAACTCCAAGAGATGGCGCCAGGTCCAAGAGGCATCAGAAATTTACACATTTCTTACCT  V  A  L  N  K  D  G  T  P  R  D  G  A  R  S  K  R  H  Q  K  F  T  H  F  L  P481 AGACCAGTGGATCCAGAAAGAGTTCCAGAATTGTACAAGGACCTACTGATGTACACTTAGR  P  V  D  P  E  R  V  P  E  L  Y  K  D  L  L  M  Y  T

[0484] TABLE 41 View DNA Sequence Analysis of 259059629 TranslatedProtein - Frame: 2 - Nucleotide 2 to 415 1CACCAGATCTATCCTGCGCCGCCGGCAGCTCTATTGCCGCACCGGCTTCCACCTGCAGATCCTGCCCGACGGCAGCGTGC T  R  S  I  L  R  R  R  Q  L  Y  C  R  T  G  F  H  L  Q  I  L  P  D  G  S  V  Q81AGGGCACCCGGCAGGACCACAGCCTCTTCGGTATCTTGGAATTCATCAGTGTGGCAGTGGGACTGGTCAGTATTAGAGGT  G  T  R  Q  D  H  S  L  F  G  I  L  E  F  I  S  V  A  V  G  L  V  S  I  R  G161GTGGACAGTGGTCTCTATCTTGGAATGAATGACAAAGGAGAACTCTATGGATCAGAGAAACTTACTTCCGAATGCATCTTV  D  S  G  L  Y  L  G  M  N  D  K  G  E  L  Y  G  S  E  K  L  T  S  E  C  I  F241TAGGGAGCAGTTTGAAGAGAACTGGTATAACACCTATTCATCTAACATATATAAACATGGAGACACTGGCCGCAGGTATT R  E  Q  F  E  E  N  W  Y  N  T  Y  S  S  N  I  Y  K  H  G  O  T  G  R  R  Y  F321TTGTGGCACTTAACAAAGACGGAACTCCAAGAGATGGCGCCAGGTCCAAGAGGCATCAGAAATTTACACATTTCTTACCT  V  A  L  N  K  D  G  T  P  R  O  G  A  R  S  K  R  H  Q  K  F  T  H  F  L  P401 AGACCAGTCGACGGC R  P  V  D  G

[0485]

[0486] C. Molecular Cloning of CG53135-04 (31-179 aa)

[0487] The cDNA coding for the mature form of CG53135-04 from residue 31to 179 was targeted for “in-frame” cloning by PCR. The PCR template isbased on the previously identified plasmid.

[0488] The following oligonucleotide primers were used to clone thetarget cDNA sequence: (SEQ ID NO:63) F2 5′-CACCAGATCTATCCTGCGCCGCCGGCAGCTCTATTGCC-3′ (SEQ ID NO:56) R1 5′-GCCGTCGACAGTGTACATCAGTAGGTCCTTGTACAATTC-3′

[0489] For downstream cloning purposes, the forward primer includes anin-frame Bgl II restriction site and the reverse primer contains anin-frame Sal I restriction site.

[0490] Two PCR reactions were set up using a total of 1-5 ng of theplasmid that contains the insert for CG53135-04.

[0491] The reaction mixtures contained 2 microliters of each of theprimers (original concentration: 5 pmol/ul), 1 microliter of 10 mM dNTP(Clontech Laboratories, Palo Alto Calif.) and 1 microliter of50×Advantage-HF 2 polymerase (Clontech Laboratories) in 50microliter-reaction volume. The reaction conditions used are provided inabove in Example 22A.

[0492] An amplified product was detected by agarose gel electrophoresis.The fragment was gel-purified and ligated into the pCR2.1 TOPO vector(Invitrogen, Carlsbad, Calif.) following the manufacturer'srecommendation. Twelve clones per PCR reaction were picked andsequenced. The inserts were sequenced using vector-specific M13 Forwardand M13 Reverse primers.

[0493] The insert assembly 250059669 was found to encode an open readingframe between residues 31 and 179 of the target sequence of CG53135-04.The cloned inserts are 100% identical to the original sequence. SeeTables 43-46 The alignment with CG53135-04 is displayed in a ClustalW inTable 47. The first 3 and the last 3 amino acid residues of theassemblies are derived from the restriction enzyme sites added in theprimers for the purpose of sub-cloning. Note that differing amino acidshave a white or grey background, and deleted/inserted amino acids can bedetected by a dashed line in the sequence that does not code at thatposition. TABLE 43 Cloned Sequences >CG53135-04ATGGCTCCCTTAGCCGAAGTCGGGGGCTTTCTGGGCGGCCTGGAGGGCTTGGGCCAGCCGGGGGCAGCGC(SEQ ID NO:67)AGCTGGCGCACCTGCACGGCATCCTGCGCCGCCGGCAGCTCTATTGCCGCACCGGCTTCCACCTGCAGATCCTGCCCGACGGCAGCGTGCAGGGCACCCGGCAGGACCACAGCCTCTTCGGTATCTTGGAATTCATCAGTGTGGCAGTGGGACTGGTCAGTATTAGAGGTGTGGACAGTGGTCTCTATCTTGGAATGAATGACAAAGGAGAACTCTATGGATCAGAGAAACTTACTTCCGAATGCATCTTTAGGGAGCAGTTTGAAGAGAACTGGTATAACACCTATTCATCTAACATATATAAACATGGAGACACTGGCCGCAGGTATTTTGTGGCACTTAACAAAGACGGAACTCCAAGAGATGGCGCCAGGTCCAAGAGGCATCAGAAATTTACACATTTCTTACCTAGACCAGTGGATCCAGAAAGAGTTCCAGAATTGTACAAGGACCTACTGATGTACACTTAG

[0494] TABLE 44 Cloned Sequences >250059669CACCAGATCTATCCTGCGCCGCCGGCAGCTCTATTGCCGCACCGGCTTCCACCTGCAGATCCTGCCCGAC(SEQ ID NO:68)GGCAGCGTGCAGGGCACCCGGCAGGACCACAGCCTCTTCGGTATCTTGGAATTCATCAGTGTGGCAGTGGGACTGGTCAGTATTAGAGGTGTGGACAGTGGTCTCTATCTTGGAATGAATGACAAAGGAGAACTCTATGGATCAGAGAAACTTACTTCCGAATGCATCTTTAGGGAGCAGTTTGAAGAGAACTGGTATAACACCTATTGATCTAACATATATAAACATGGAGACACTGGCCGCAGGTATTTTGTGGCACTTAACAAAGACGGAACTCCAAGAGATGGCGCCAGGTCCAAGAGGCATCAGAAATTTACACATTTCTTACCTAGACCAGTGGATCCAGAAAGAGTTCCAGAATTGTACAAGGACCTACTGATGTACACTGTCGACGGC

[0495] TABLE 45 View DNA Sequence Analysis of CG53135-04 TranslatedProtein - Frame: 1 - Nucleotide 1 to 537 1ATGGCTCCCTTAGCCGAAGTCGGGGGCTTTCTGGGCGGCCTGGAGGGCTTGGGCCAGCCGGGGGCAGCGCAGCTGGCGCAM  A  P  L  A  E  V  G  G  F  L  G  G  L  E  G  L  G  Q  P  G  A  A  Q  L  A  H81CCTGCACGGCATCCTGCGCCGCCGGCAGCTCTATTGCCGCACCGGCTTCCACCTGCAGATCCTGCCCGACGGCAGCGTGC L  H  G  I  L  R  R  R  Q  L  Y  C  R  T  G  F  H  L  Q  I  L  P  D  G  S  V  Q161AGGGCACCCGGCAGGACCACAGCCTCTTCGGTATCTTGGAATTCATCAGTGTGGCAGTGGGACTGGTCAGTATTAGAGGT  G  T  R  Q  D  H  S  L  F  G  I  L  E  F  I  S  V  A  V  G  L  V  S  I  R  G241GTGGACAGTGGTCTCTATCTTGGAATGAATGACAAAGGAGAACTCTATGGATCAGAGAAACTTACTTCCGAATGCATCTTV  D  S  G  L  Y  L  G  M  N  D  K  G  E  L  Y  G  S  H  K  L  T  S  E  C  I  F321TAGGGAGCAGTTTGAAGAGAACTGGTATAACACCTATTCATCTAACATATATAAACATGGAGACACTGGCCGCAGGTATT R  E  Q  F  E  E  N  W  Y  N  T  Y  S  S  N  I  Y  K  H  G  D  T  G  R  R  Y  F401TTGTGGCACTTAACAAAGACGGAACTCCAAGAGATGGCGCCAGGTCCAAGAGGCATCAGAAATTTACACATTTCTTACCT  V  A  L  N  K  D  G  T  P  R  D  G  A  R  S  K  R  H  Q  K  F  T  H  F  L  P481 AGACCAGTGGATCCAGAAAGAGTTCCAGAATTGTACAAGGACCTACTGATGTACACTTAGR  P  V  D  P  E  R  V  P  E  L  Y  K  D  L  L  M  Y  T+TZ,53

[0496] TABLE 46 View DNA Sequence Analysis of 250059669 TranslatedProtein - Frame: 2 - Nucleotide 2 to 466 1CACCAGATCTATCCTGCGCCGCCGGCAGCTCTATTGCCGCACCGGCTTCCACCTGCAGATCCTGCCCGACGGCAGCGTGC T  R  S  I  L  R  R  R  Q  L  Y  C  R  T  G  F  H  L  Q  I  L  P  D  G  S  V  Q81AGGGCACCCGGCAGGACCACAGCCTCTTCGGTATCTTGGAATTCATCAGTGTGGCAGTGGGACTGGTCAGTATTAGAGGT  G  T  R  Q  D  H  S  L  F  G  I  L  H  F  I  S  V  A  V  G  L  V  S  I  R  G161GTGGACAGTGGTCTCTATCTTGGAATGAATGACAAAGGAGAACTCTATGGATCAGAGAAACTTACTICCGAATGCATCTTV  D  S  G  L  Y  L  G  M  N  D  K  G  E  L  Y  G  S  E  K  L  T  S  E  C  I  F241TAGGGAGCAGTTTGAAGAGAACTGGTATAACACCTATTCATCTAACATATATAAACATGGAGACACTGGCCGCAGGTATT R  E  Q  F  E  E  N  W  Y  N  T  Y  S  S  N  I  Y  K  H  G  D  T  G  R  R  Y  F321TTGTGGCACTTAACAAAGACGGAACTCCAAGAGATGGCGCCAGGTCCAAGAGGCATCAGAAATTTACACATTTCTTACCT  V  A  L  N  K  D  G  F  P  R  D  G  A  R  S  K  R  H  Q  K  F  T  H  F  L  P401 AGACCAGTGGATCCAGAAAGAGTTCCAGAATTGTACAAGGACCTACTGATGTACACTGTCGACGGCR  P  V  D  P  H  R  V  P  H  L  Y  K  D  L  L  M  Y  T  V  D  G

[0497]

Equivalents

[0498] From the foregoing detailed description of the specificembodiments of the invention, it should be apparent that particularnovel compositions and methods involving nucleic acids, polypeptides,antibodies, detection and treatment have been described. Although theseparticular embodiments have been disclosed herein in detail, this hasbeen done by way of example for purposes of illustration only, and isnot intended to be limiting with respect to the scope of the appendedclaims that follow. In particular, it is contemplated by the inventorsthat various substitutions, alterations, and modifications may be madeas a matter of routine for a person of ordinary skill in the art to theinvention without departing from the spirit and scope of the inventionas defined by the claims. Indeed, various modifications of the inventionin addition to those described herein will become apparent to thoseskilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

What is claimed is:
 1. An isolated polypeptide comprising an amino acidsequence selected from the group consisting of: a) an amino acidsequence given by SEQ ID NO: 2; b) a variant of an amino acid sequencegiven by SEQ ID NO: 2, in which any amino acid specified in the chosensequence is changed to a different amino acid, provided that no morethan 15% of the amino acid residues in the sequence are so changed; c) amature form of an amino acid sequence given by SEQ ID NO: 2; and d) avariant of a mature form of an amino acid sequence given by SEQ ID NO:2, wherein any amino acid in the mature form of the chosen sequence ischanged to a different amino acid, provided that no more than 15% of theamino acid residues in the sequence of the mature form are so changed;and e) a fragment of an amino acid sequence described in paragraphs a)to d).
 2. A fragment of a polypeptide described in claim
 1. 3. Thepolypeptide of claim 1, wherein said polypeptide is a naturallyoccurring allelic variant of SEQ ID NO:
 2. 4. The polypeptide of claim3, wherein the variant is the translation of a single nucleotidepolymorphism.
 5. The polypeptide of claim 1, wherein said polypeptide isa variant polypeptide, and wherein one or more of any amino acidspecified in SEQ ID NO: 2 is changed to provide a conservativesubstitution.
 6. An isolated nucleic acid molecule comprising a nucleicacid sequence encoding a polypeptide comprising an amino acid sequenceselected from the group consisting of: a) a polypeptide comprising SEQID NO: 2; b) a variant of SEQ ID NO: 2, in which any amino acidspecified in the chosen sequence is changed to a different amino acid,provided that no more than 15% of the amino acid residues in thesequence are so changed; c) a mature form of the amino acid sequencegiven by SEQ ID NO: 2; and d) a variant of a mature form of the aminoacid sequence given by SEQ ID NO: 2, wherein any amino acid in themature form of the chosen sequence is changed to a different amino acid,provided that no more than 15% of the amino acid residues in thesequence of the mature form are so changed; e) a fragment of an aminoacid sequence described in a) to d); and f) the complement of any of thenucleic acid molecules described in paragraphs a) to e).
 7. The nucleicacid molecule of claim 6, wherein the nucleic acid molecule comprisesthe nucleotide sequence of a naturally occurring allelic nucleic acidvariant.
 8. The nucleic acid molecule of claim 6, wherein said nucleicacid molecule encodes a variant polypeptide that has the polypeptidesequence of a naturally occurring polypeptide variant.
 9. The nucleicacid molecule of claim 6, wherein the nucleic acid molecule comprises asingle nucleotide polymorphism encoding said variant polypeptide. 10.The nucleic acid molecule of claim 6, wherein said nucleic acid moleculecomprises a nucleotide sequence selected from the group consisting of a)a nucleotide sequence given by SEQ ID NO: 1; b) a nucleotide sequencewherein one or more nucleotides in a nucleotide sequence given by SEQ IDNO: 1 is changed from that given by the chosen sequence to a differentnucleotide provided that no more than 20% of the nucleotides are sochanged; c) a nucleic acid fragment of the sequence described in a); d)a nucleic acid fragment of the sequence described in b); and e) thecomplement of any of said nucleic acid molecules.
 11. The nucleic acidmolecule of claim 6, wherein the nucleic acid molecule comprises anucleotide sequence in which any nucleotide specified in the codingsequence of the chosen nucleotide sequence is changed from that given bythe chosen sequence to a different nucleotide provided that no more than20% of the nucleotides in the chosen coding sequence are so changed. 12.An isolated nucleic acid molecule encoding a fragment of an FGF-CXpolypeptide.
 13. The nucleic acid molecule of claim 12, wherein saidFGF-CX polypeptide is a variant of SEQ ID NO:
 2. 14. The nucleic acidmolecule of claim 12,wherein said FGF-CX polypeptide is a mature FGF-CXpolypeptide.
 15. The nucleic acid molecule of claim 12, wherein saidFGF-CX polypeptide is a variant of a mature form of SEQ ID NO:
 2. 16. Avector comprising the nucleic acid molecule of claim
 6. 17. A cellcomprising the vector of claim
 16. 18. An antibody that bindsimmunospecifically to an isolated polypeptide comprising an amino acidsequence selected from the group consisting of: (a) an amino acidsequence given by SEQ ID NO: 2; (b) a variant of an amino acid sequencegiven by SEQ ID NO: 2, in which any amino acid specified in the chosensequence is changed to a different amino acid, provided that no morethan 15% of the amino acid residues in the sequence are so changed; (c)a mature form of an amino acid sequence given by SEQ ID NO: 2; and (d) avariant of a mature form of an amino acid sequence given by SEQ ID NO:2, wherein any amino acid in the mature form of the chosen sequence ischanged to a different amino acid, provided that no more than 15% of theamino acid residues in the sequence of the mature form are so changed;and (e) a fragment of an amino acid sequence described in paragraphs (a)to (d).
 19. The antibody of claim 18, wherein said antibody is amonoclonal antibody.
 20. The antibody of claim 18, wherein the antibodyis a humanized antibody or a human antibody.
 21. A method fordetermining the presence or amount of an isolated polypeptide comprisingan amino acid sequence selected from the group consisting of: (a) anamino acid sequence given by SEQ ID NO: 2; (b) a variant of an aminoacid sequence given by SEQ ID NO: 2, in which any amino acid specifiedin the chosen sequence is changed to a different amino acid, providedthat no more than 15% of the amino acid residues in the sequence are sochanged; (c) a mature form of an amino acid sequence given by SEQ ID NO:2; and (d) a variant of a mature form of an amino acid sequence given bySEQ ID NO: 2, wherein any amino acid in the mature form of the chosensequence is changed to a different amino acid, provided that no morethan 15% of the amino acid residues in the sequence of the mature formare so changed; and (e) a fragment of an amino acid sequence describedin paragraphs (a) to (d); in a sample, the method comprising: (i)providing the sample; (ii) contacting the sample with an antibody thatbinds immunospecifically to the polypeptide; and (iii) determining thepresence or amount of antibody bound to said polypeptide, therebydetermining the presence or amount of polypeptide in said sample.
 22. Amethod for determining the presence or amount of a nucleic acid moleculeof claim 6 in a sample, the method comprising: (i) providing the sample;(ii) contacting the sample with a probe that binds to said nucleic acidmolecule; and (iii) determining the presence or amount of the probebound to said nucleic acid molecule, thereby determining the presence oramount of the nucleic acid molecule in said sample.
 23. A method foridentifying an agent that binds to an isolated polypeptide comprising anamino acid sequence selected from the group consisting of: (a) an aminoacid sequence given by SEQ ID NO: 2; (b) a variant of an amino acidsequence given by SEQ ID NO: 2, in which any amino acid specified in thechosen sequence is changed to a different amino acid, provided that nomore than 15% of the amino acid residues in the sequence are so changed;(c) a mature form of an amino acid sequence given by SEQ ID NO: 2; and(d) a variant of a mature form of an amino acid sequence given by SEQ IDNO: 2, wherein any amino acid in the mature form of the chosen sequenceis changed to a different amino acid, provided that no more than 15% ofthe amino acid residues in the sequence of the mature form are sochanged; and (e) a fragment of an amino acid sequence described inparagraphs (a) to (d), the method comprising: (i) contacting saidpolypeptide with a candidate substance; and (i) determining whether saidcandidate substance binds to said polypeptide; wherein a candidatesubstance that binds is the agent.
 24. The method of claim 23 whereinthe candidate substance has a molecular weight not more than about 1500Da.
 25. A method for modulating an activity of an isolated polypeptidecomprising an amino acid sequence selected from the group consisting of:(a) an amino acid sequence given by SEQ ID NO: 2; (b) a variant of anamino acid sequence given by SEQ ID NO: 2, in which any amino acidspecified in the chosen sequence is changed to a different amino acid,provided that no more than 15% of the amino acid residues in thesequence are so changed; (c) a mature form of an amino acid sequencegiven by SEQ ID NO: 2; and (d) a variant of a mature form of an aminoacid sequence given by SEQ ID NO: 2, wherein any amino acid in themature form of the chosen sequence is changed to a different amino acid,provided that no more than 15% of the amino acid residues in thesequence of the mature form are so changed; and (e) a fragment of anamino acid sequence described in paragraphs (a) to (d), the methodcomprising contacting the polypeptide with a compound that binds to thepolypeptide in an amount sufficient to modulate the activity of thepolypeptide.
 26. A method for identifying a potential therapeutic agentfor use in treatment of a pathology, wherein the pathology is related toaberrant expression, aberrant processing, or aberrant physiologicalinteractions of an isolated polypeptide comprising an amino acidsequence selected from the group consisting of: (a) an amino acidsequence given by SEQ ID NO: 2; (b) a variant of an amino acid sequencegiven by SEQ ID NO: 2, in which any amino acid specified in the chosensequence is changed to a different amino acid, provided that no morethan 15% of the amino acid residues in the sequence are so changed; (c)a mature form of an amino acid sequence given by SEQ ID NO: 2; and (d) avariant of a mature form of an amino acid sequence given by SEQ ID NO:2, wherein any amino acid in the mature form of the chosen sequence ischanged to a different amino acid, provided that no more than 15% of theamino acid residues in the sequence of the mature form are so changed;and (e) a fragment of an amino acid sequence described in paragraphs (a)to (d), the method comprising: (i) providing a cell expressing thepolypeptide and having a property or function ascribable to thepolypeptide; (ii) contacting the cell provided in step (a) with a testagent; and (iii) determining whether the test agent alters the propertyor function ascribable to the polypeptide; whereby an alteration of theproperty or function of the polypeptide observed in the presence of thetest agent indicates the test agent is a potential therapeutic agent.27. The method of claim 26, further comprising subjecting the potentialtherapeutic agent to additional tests to identify the therapeutic agent.28. The method of claim 26, wherein the candidate substance is anantibody or has a molecular weight not more than about 1500 Da.
 29. Themethod of claim 26, wherein the property or function comprises cellgrowth or cell proliferation.
 30. The method of claim 29, wherein thetest agent binds to the polypeptide.
 31. A therapeutic agent identifiedaccording to the method of claim
 26. 32. A therapeutic agent identifiedusing the method of claim
 27. 33. The therapeutic agent of claim 31,wherein the agent is an antibody or has a molecular weight not more thanabout 1500 Da.
 34. A method of treating or preventing a disorderassociated with an isolated polypeptide comprising an amino acidsequence; said amino acid sequence selected from the group consistingof: (a) an amino acid sequence given by SEQ ID NO: 2; (b) a variant ofan amino acid sequence given by SEQ ID NO: 2, in which any amino acidspecified in the chosen sequence is changed to a different amino acid,provided that no more than 15% of the amino acid residues in thesequence are so changed; (c) a mature form of an amino acid sequencegiven by SEQ ID NO: 2; and (d) a variant of a mature form of an aminoacid sequence given by SEQ ID NO: 2, wherein any amino acid in themature form of the chosen sequence is changed to a different amino acid,provided that no more than 15% of the amino acid residues in thesequence of the mature form are so changed; and (e) a fragment of anamino acid sequence described in paragraphs (a) to (d); wherein thedisorder is characterized by insufficient or ineffective growth of acell or a tissue, said method comprising administering to a subject saidpolypeptide in an amount and for a duration sufficient to treat orprevent said polypeptide-associated disorder in said subject, whereinthe subject is thought to be prone to or to be suffering from thedisorder.
 35. The method of claim 34, wherein said subject is a human.36. A method of treating or preventing a disorder associated withaberrant expression, aberrant processing, or aberrant physiologicalinteractions of: an isolated polypeptide comprising an amino acidsequence selected from the group consisting of: (a) an amino acidsequence given by SEQ ID NO: 2; (b) a variant of an amino acid sequencegiven by SEQ ID NO: 2, in which any amino acid specified in the chosensequence is changed to a different amino acid, provided that no morethan 15% of the amino acid residues in the sequence are so changed; (c)a mature form of an amino acid sequence given by SEQ ID NO: 2; and (d) avariant of a mature form of an amino acid sequence given by SEQ ID NO:2, wherein any amino acid in the mature form of the chosen sequence ischanged to a different amino acid, provided that no more than 15% of theamino acid residues in the sequence of the mature form are so changed;and (e) a fragment of an amino acid sequence described in paragraphs a)to d); wherein the disorder is characterized by insufficient orineffective growth of a cell or a tissue, said method comprisingadministering to a subject an isolated nucleic acid molecule comprisinga nucleic acid sequence encoding a polypeptide comprising an amino acidsequence selected from the group consisting of: (e) a polypeptidecomprising SEQ ID NO: 2; (f) a variant of SEQ ID NO: 2, in which anyamino acid specified in the chosen sequence is changed to a differentamino acid, provided that no more than 15% of the amino acid residues inthe sequence are so changed; (g) a mature form of the amino acidsequence given by SEQ ID NO: 2; and (h) a variant of a mature form ofthe amino acid sequence given by SEQ ID NO: 2, wherein any amino acid inthe mature form of the chosen sequence is changed to a different aminoacid, provided that no more than 15% of the amino acid residues in thesequence of the mature form are so changed; (i) a fragment of an aminoacid sequence described in (e) to (h); and 0) the complement of any ofthe nucleic acid molecules described in paragraphs (e) to (i); in anamount and for a duration sufficient to treat or prevent said disorderin said subject, wherein the subject is thought to be prone to or to besuffering from the disorder.
 37. The method of claim 36, wherein saidsubject is a human.
 38. A method of treating or preventing a disorderassociated with aberrant expression, aberrant processing, or aberrantphysiological interactions of a polypeptide described in claim 1,wherein the disorder is characterized by hyperplasia or neoplasia of acell or a tissue, said method comprising administering to a subject aTherapeutic in an amount sufficient to treat or prevent said disorder insaid subject, wherein the subject is thought to be prone to or to besuffering from the disorder.
 39. The method described in claim 38wherein the Therapeutic is an antibody against said polypeptide.
 40. Themethod of claim 38, wherein the subject is a human.
 41. A pharmaceuticalcomposition comprising the polypeptide of claim 1 and a pharmaceuticallyacceptable carrier.
 42. A pharmaceutical composition comprising thenucleic acid molecule of claim 6 and a pharmaceutically acceptablecarrier.
 43. A pharmaceutical composition comprising the antibody ofclaim 18 and a pharmaceutically acceptable carrier.
 44. A pharmaceuticalcomposition comprising the therapeutic agent of claim 31 and apharmaceutically acceptable carrier.
 45. The pharmaceutical compositionof claim 44, wherein the therapeutic agent has a molecular weight notmore than about 1500 Da.
 46. A kit comprising in one or more containersa pharmaceutical composition of claim
 41. 47. A kit comprising in one ormore containers a pharmaceutical composition of claim
 42. 48. A kitcomprising in one or more containers a pharmaceutical composition ofclaim
 43. 49. A method for screening for a modulator of latency orpredisposition to a disorder associated with aberrant expression,aberrant processing, or aberrant physiological interactions of apolypeptide described in claim 1, said method comprising: a) providing atest animal at increased risk for the disorder and wherein said testanimal recombinantly expresses the polypeptide of claim 1; b)administering a test compound to the test animal; c) measuring anactivity of said polypeptide in said test animal after administering thecompound of step (a);and d) comparing the activity of said protein insaid test animal with the activity of said polypeptide in a controlanimal not administered said compound; wherein a change in the activityof said polypeptide in said test animal relative to said control animalindicates the test compound is a modulator of latency of orpredisposition to the disorder.
 50. The method of claim 49, wherein saidtest animal is a recombinant test animal that expresses a test proteintransgene or expresses said transgene under the control of a promoter atan increased level relative to a wild-type test animal, and wherein saidpromoter is not the native gene promoter of said transgene.
 51. A methodfor determining the presence of or predisposition to a diseaseassociated with altered levels of a polypeptide described in claim 1 ina first mammalian subject, the method comprising: a) measuring the levelof expression of the polypeptide in a sample from the first mammaliansubject; and b) comparing the amount of said polypeptide in the sampleof step (a) to the amount of the polypeptide present in a control samplefrom a second mammalian subject known not to have, or not to bepredisposed to, said disease, wherein an alteration in the expressionlevel of the polypeptide in the first subject as compared to the controlsample indicates the presence of or predisposition to said disease. 52.A method for determining the presence of or predisposition to a diseaseassociated with altered levels of a nucleic acid molecule described inclaim 6 in a first mammalian subject, the method comprising: a)measuring the amount of the nucleic acid in a sample from the firstmammalian subject; and b) comparing the amount of said nucleic acid inthe sample of step (a) to the amount of the nucleic acid present in acontrol sample from a second mammalian subject known not to have or notbe predisposed to, the disease; wherein an alteration in the level ofthe nucleic acid in the first subject as compared to the control sampleindicates the presence of or predisposition to the disease.
 53. A methodof treating a pathological state in a mammal, wherein the pathology isrelated to aberrant expression, aberrant processing, or aberrantphysiological interactions of a polypeptide described in claim 1, themethod comprising administering to the mammal a polypeptide in an amountthat is sufficient to alleviate the pathological state, wherein thepolypeptide is a polypeptide having an amino acid sequence at least 95%identical to a polypeptide comprising an amino acid sequence of SEQ IDNO: 2, or a biologically active fragment thereof.
 54. A method oftreating a pathological state in a mammal, wherein the pathology isrelated to aberrant expression, aberrant processing, or aberrantphysiological interactions of an FGF-CX polypeptide, the methodcomprising administering to the mammal an antibody described in claim 18in an amount and for a duration sufficient to alleviate the pathologicalstate.
 55. A method of promoting growth of cells in a subject comprisingadministering to a subject in need thereof a polypeptide described inclaim 1 in an amount and for a duration that are effective to promotecell growth.
 56. The method of claim 55, wherein the subject is a human.57. The method described in claim 55 wherein the cells whose growth isto be promoted are chosen from the group consisting of cells in thevicinity of a wound, cells in the vascular system, cells involved inhematopoiesis, cells involved in erythropoiesis, cells in the lining ofthe gastrointestinal tract, and cells in hair follicles.
 58. A method ofinhibiting growth of cells in a subject, wherein the growth is relatedto expression of a polypeptide described in claim 1, comprisingadministering to the subject a composition in an amount sufficient toinhibit growth of cells in said subject.
 59. The method of claim 58,wherein the composition inhibits the cleavage of an FGF-CX polypeptide.60. The method of claim 58, wherein the composition comprises an antFGF-CX antibody or a FGF-CX therapeutic agent.
 61. The method of claim58, wherein the subject is a human.
 62. The method of claim 58, whereinthe cells whose growth is to be inhibited are chosen from the groupconsisting of transformed cells, hyperplastic cells, tumor cells, andneoplastic cells.
 63. The polypeptide fragment described in claim 2,wherein the fragment comprises an amino acid sequence selected from thegroup consisting of residues 55-211 of SEQ ID NO: 2 and residues 24-211of SEQ ID NO:
 2. 64. The isolated nucleic acid molecule described inclaim 6, wherein the nucleic acid molecule comprises a nucleic acidsequence encoding a polypeptide fragment comprising an amino acidsequence selected from the group consisting of residues 55-211 of SEQ IDNO: 2 and residues 24-211 of SEQ ID NO:
 2. 65. The nucleic acid moleculedescribed in claim 10, wherein the nucleic acid sequence comprises asequence selected from the group consisting of nucleotides 163-633 ofSEQ ID NO: 1 and nucleotides 70-633 of SEQ ID NO: 1.